Optimized human coagulation factor VIII gene expression cassette and its use

Abstract

The present invention relates to an optimized human coagulation factor VIII gene expression cassette and its use, in particular to a synthetic liver-specific promoter and expression construct for producing polypeptides and functional nucleic acids in the liver of a subject. The present invention also relates to a factor VIII protein containing a modification in the amino acid sequence of the factor VIII protein, as well as a nucleic acid construct encoding the factor VIII protein and a method of using these compositions to treat bleeding disorders.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680020293.7, filed on February 5, 2016, entitled “Optimized Human Coagulation Factor VIII Gene Expression Cassette and Uses Thereof”.

[0002] Priority declaration

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 112,901, filed February 6, 2015, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present invention relates to synthetic liver-specific promoters and expression constructs for producing polypeptides and functional nucleic acids in the liver of a subject. The present invention also relates to Factor VIII proteins containing modifications in the amino acid sequence of the Factor VIII protein, as well as nucleic acid constructs encoding the Factor VIII protein and methods of using these compositions to treat hemorrhagic disorders. Background Art

[0005] Factor VIII (FVIII) plays a key role in the coagulation cascade by accelerating the conversion of factor X into factor Xa. Lack of FVIII activity is the cause of the bleeding disorder hemophilia A. The current treatment for hemophilia A is intravenous infusion of plasma-derived or recombinant FVIII protein. Although this treatment can effectively control bleeding episodes, due to the short half-life of FVIII (8-12 hours), the requirement for frequent infusion makes it inherently costly. Gene therapy has become an attractive strategy for the ultimate cure of this disease. However, the progress of delivering the FVIII gene using adeno-associated virus (AAV), one of the most promising viral vectors, has lagged behind the progress of delivering coagulation factor IX, because the large size of the FVIII coding sequence is close to the packaging capacity of AAV.

[0006] The present invention overcomes the shortcomings of the art by providing a short synthetic liver-specific promoter and expression construct suitable for use in an AAV vector. The present invention further provides a FVIII protein comprising an additional glycosylation site with an amino acid sequence modification and a method for using the same for treating a bleeding disorder. Summary of the invention

[0007] The present invention is based in part on the development of a synthetic liver-specific promoter that is only about 200 base pairs long. The promoter can be used to produce polypeptides and functional nucleic acids in a liver-specific manner, especially using AAV vectors, which have strict length restrictions and can benefit from the availability of short but strong promoters.

[0008] The present invention is also based in part on the development of modified FVIII proteins comprising additional glycosylation sites in the heavy chain. The modified proteins provide prolonged and high levels of activity relative to FVIII proteins without the modifications described herein.

[0009] In one aspect, the present invention relates to a polynucleotide comprising a synthetic liver-specific promoter, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0010] In another aspect, the present invention relates to a vector, a cell and / or a transgenic animal comprising a polynucleotide of the present invention.

[0011] In another aspect, the present invention relates to a method for producing a polypeptide or a functional nucleic acid in the liver of a subject, comprising delivering the polynucleotide, vector and / or transformed cell of the present invention to the subject, thereby producing the polypeptide or the functional nucleic acid in the liver of the subject.

[0012] In an additional aspect, the invention relates to a method of treating hemophilia A in a subject, comprising delivering to the subject a therapeutically effective amount of a polynucleotide, vector and / or transformed cell of the invention, thereby treating hemophilia A in the subject.

[0013] In another aspect, the present invention relates to a method of increasing the bioavailability of a Factor VIII polypeptide in a subject, comprising delivering to the subject an effective amount of a polynucleotide, vector and / or transformed cell of the present invention, thereby increasing the bioavailability of the Factor VIII polypeptide in the subject.

[0014] In yet another aspect, the invention relates to a modified human Factor VIII polypeptide, wherein the amino acid residues in the heavy chain are modified to generate one or more glycosylation sites.

[0015] In additional aspects, the present invention relates to a polynucleotide encoding a modified human Factor VIII polypeptide of the present invention and a vector, a cell and / or a transgenic animal comprising the polynucleotide.

[0016] In another aspect, the present invention relates to a method for producing Factor VIII in the liver of a subject, comprising delivering to the subject a polynucleotide encoding a modified human Factor VIII polypeptide of the present invention, or a vector and / or transformed cells comprising the polynucleotide, thereby producing Factor VIII in the liver of the subject.

[0017] In another aspect, the invention relates to a method for treating hemophilia A in a subject, comprising delivering to the subject a therapeutically effective amount of a modified human Factor VIII polypeptide, polynucleotide, vector and / or transformed cell of the invention, thereby treating hemophilia A in the subject.

[0018] In an additional aspect, the present invention relates to a method for increasing the bioavailability of a Factor VIII polypeptide in a subject, comprising delivering to the subject an effective amount of a polynucleotide encoding a modified human Factor VIII polypeptide of the present invention, or a vector and / or transformed cells comprising the polynucleotide, thereby increasing the bioavailability of the Factor VIII polypeptide in the subject.

[0019] These and other aspects of the invention are set forth in more detail in the description of the invention which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The sequence of the LXP3.3 promoter is shown (SEQ ID NO: 1). Putative liver and housekeeping transcription factor binding sites are highlighted by underlining.

[0021] Figure 2A Comparison of LacZ expression in liver and heart following intravenous injection of AAV9-TBG-LacZ or AAV9-Lxp3.3-LacZ into mice is shown. Shown are X-gal and H&E double staining of liver and heart thin sections.

[0022] Figure 2B Shown in use 1x10 11 Quantitative comparison of LacZ enzyme activity in various tissues of mice treated with vector genomes (vg) of AAV9-LacZ vectors containing either the nonspecific CMV promoter, the liver-specific TBG promoter, or the LXP3.3 promoter (e.g. Figure 1 shown).

[0023] Figure 2C Shows the use of Figure 2B The AAV9-LacZ vectors shown above were used but at a lower dose (2x10 10 Quantitative comparison of LacZ enzyme activity in the livers of mice treated with 10 vector genomes (vg).

[0024] Figure 3 Shown are Factor VIII activities in supernatants of Huh7 cells transfected with different AAV vector plasmids containing the NBP promoter (177 bp) driving the human BDD Factor VIII gene without introns or containing the VH4 intron or the chimeric CIN intron.

[0025] Figure 4The sequence of the AAV-Lxp3.3i-BDD-F8 construct and the Lxp3.3i promoter-intron is shown (SEQ ID NO: 2). ITR stands for the 145 bp inverted terminal repeat sequence of AAV.

[0026] Figure 5 Shown is the expression of FVIII in human hepatoma Huh7 cells and mouse Hepa1-6 cells for two constructs containing either the nonspecific CMV promoter or the liver-specific LXP3.3 promoter.

[0027] Figure 6 Shown are FVIII activities in Huh7 cells transfected with different BDD Factor VIII (synthetic opti-F8 or wild-type wtF8) plasmids containing the promoter LXP3.3 or the weak promoter TkPro, respectively.

[0028] Fig. 7A showed that high doses (2x10 11 vg / mouse) or low dose (4x10 10 AAV9-Lxp3.3-F8-mediated long-term human FVIII gene expression and FVIII activity in FVIII knockout mice after 4 vg / mouse (Figure 5A). Factor VIII activity (as a percentage of normal human levels) was measured at 2, 6, 10, and 18 weeks after injection.

[0029] Figure 7B showed that high doses (2x10 11 vg / mouse) or low dose (4x10 10 Long-term human FVIII gene expression and human FVIII protein concentration mediated by AAV9-Lxp3.3-F8 in FVIII knockout mice after 4 weeks of injection (vg / mouse). Factor VIII protein (as a percentage of normal human levels) was measured by ELISA at 2, 6, 10 and 18 weeks after injection.

[0030] Figure 8 The amino acid sequences of modified BDD FVIII proteins are shown (SEQ ID NOs: 31-34). SQ represents Serine 743 (Ser 743) and Glutamine (Gln 1638) at the junction of the heavy and light chains of BDD Factor VIII (numbering based on SEQ ID NO: 5). The underlined letters highlight the mutated amino acids in the heavy chain.

[0031] Fig.9AHuman FVIII activity in Huh7 cells transfected with expression plasmids containing BDD Factor VIII or mutants F8X1 and F8X2 is shown (see Figure 8 ).

[0032] Fig. 9B Long-term AAV8 vector-mediated expression of the mutant human BDD FVIII gene in FVIII knockout mice is shown. Human factor VIII activity (as a percentage of normal human levels) was increased following intravenous injection of 5x10 10 The measurements were performed at different time points after addition of each AAV8-LXP3.3i-F8X1 or AAV8-LXP3.3i-F8X2 vector genome. DETAILED DESCRIPTION

[0033] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the present invention are shown. However, the present invention may be implemented in different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art.

[0034] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein may be used in any combination. In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted. For illustration, if the specification states that a composite comprises components A, B, and C, it is specifically intended that any one or combination of A, B, or C may be omitted and abandoned, either alone or in any combination.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to limit the invention.

[0036] Unless otherwise expressly indicated, nucleotide sequences are presented herein only as a single strand in the 5' to 3' direction from left to right. Nucleotides and amino acids are referred to herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids) by the single-letter code or the three-letter code, both in accordance with 37 C.FR §1.822 and established usage.

[0037] Unless otherwise indicated, standard methods known to those skilled in the art can be used for cloning genes, amplifying and detecting nucleic acids, etc. These techniques are known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor, NY, 1989); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0038] definition

[0039] As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0040] Furthermore, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted as an alternative ("or").

[0041] As used herein, the term "about" when referring to a measurable value such as an amount of a polypeptide, dosage, time, temperature, enzyme activity or other biological activity, etc., is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the specified amount.

[0042] The transitional phrase “consisting essentially of” means that the scope of the claim should be interpreted to include the specified materials or steps, and will not essentially Affecting the claimed invention Basics and novel Those of the characteristics of the invention." See In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis original); see also MPEP §2111.03.

[0043] The term "consisting essentially of..." (and grammatical variants) applied to the polynucleotide or polypeptide sequence of the present invention refers to a polynucleotide or polypeptide consisting of the sequence (e.g., SEQ ID NO) and a total of ten or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids at the 5' and / or 3' or N-terminus and / or C-terminus of the sequence, so that the function of the polynucleotide or polypeptide is not substantially changed. The total number of ten or fewer additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added at both ends. The term "substantially altered" applied to the polynucleotide of the present invention refers to an increase or decrease in the ability to express the encoded polypeptide by at least about 50% or more compared to the expression level of the polynucleotide consisting of the sequence. The term "substantially altered" applied to the polypeptide of the present invention refers to an increase or decrease in coagulation stimulating activity by at least about 50% or more compared to the activity of the polypeptide consisting of the sequence.

[0044] As used herein, the terms "enhance" or "increase" or grammatical variations thereof, refer to an increase in a specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold or even 15-fold.

[0045] As used herein, the term "inhibit" or "reduce" or grammatical variations thereof refers to a decrease or reduction in a specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In specific embodiments, the inhibition or reduction results in little or substantially undetectable activity (at most an insignificant amount, e.g., less than about 10% or even 5%).

[0046] As used herein, an "effective" amount is an amount that provides the desired effect.

[0047] As used herein, a "therapeutically effective" amount is an amount that provides some improvement or benefit to a subject. Alternatively, a "therapeutically effective" amount is an amount that will provide some relief, alleviation, or reduction in at least one clinical symptom in a subject. One skilled in the art will appreciate that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject.

[0048] As used herein, a "prophylactically effective" amount is an amount sufficient to prevent (as defined herein) a disease, disorder and / or clinical symptoms in a patient. One skilled in the art will appreciate that the level of prevention need not be complete as long as some benefit is provided to the subject.

[0049] As is well known to those skilled in the art, the efficacy of treating a bleeding disorder by the methods of the present invention can be determined by detecting clinical improvement indicated by changes in symptoms and / or clinical parameters in the subject.

[0050] The terms "treat," "treating," or "treatment" are intended to alleviate or at least partially improve or alter the severity of a subject's condition and to achieve some relief, alleviation, or reduction of at least one clinical symptom.

[0051] The terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to a reduction or delay in the extent or severity of a disease, disorder, and / or clinical symptom after onset relative to what would have occurred had the methods of the invention not been performed prior to onset of the disease, disorder, and / or clinical symptom. In the context of hemophilia A, "prevention" refers to a reduction in the number and / or severity of bleeding episodes compared to the number and / or severity of bleeding episodes that would have occurred in the absence of prophylactic treatment.

[0052] As used herein, "nucleic acid", "nucleotide sequence" and "polynucleotide" are used interchangeably and cover both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The terms polynucleotide, nucleotide sequence or nucleic acid refer to a chain of nucleotides regardless of chain length. Nucleic acids can be double-stranded or single-stranded. When single-stranded, the nucleic acid can be a sense strand or an antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or thiophosphate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids with altered base pairing ability or increased nuclease resistance. The present invention also provides a nucleic acid that is a complement (which can be a complete complement or a partial complement) of a nucleic acid, nucleotide sequence or polynucleotide of the present invention.

[0053] An "isolated polynucleotide" is a nucleotide sequence (e.g., DNA or RNA) that is not directly adjacent to the nucleotide sequence that is directly adjacent to it (one at the 5' end and one at the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequence that is immediately adjacent to the coding sequence. Thus, the term includes, for example, recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or recombinant DNA that exists as a separate molecule (e.g., cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) that is unrelated to other sequences. It also includes recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence. An isolated polynucleotide that includes a gene is not a fragment of a chromosome that includes such a gene, but includes coding regions and regulatory regions associated with the gene, but without the additional genes naturally present on the chromosome.

[0054] The term "fragment" applied to polynucleotides will be understood to refer to a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence, and comprising, substantially consisting of, and / or consisting of, a nucleotide sequence of continuous nucleotides that are consistent or almost consistent (e.g., 90%, 92%, 95%, 98%, 99% consistent) with the reference nucleic acid or nucleotide sequence. Such nucleic acid fragments according to the present invention may be included in larger polynucleotides as a composition under appropriate circumstances. In some embodiments, such a fragment may include, substantially consist of, and / or consist of, oligonucleotides having at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more continuous nucleotides of a nucleic acid or nucleotide sequence according to the present invention.

[0055] The term "isolated" may refer to a nucleic acid, nucleotide sequence, or polypeptide that is substantially free of cellular material, viral material, and / or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). In addition, an "isolated fragment" is a fragment of a nucleic acid, nucleotide sequence, or polypeptide that is not a naturally occurring fragment and would not be found in nature. "Isolated" does not mean that the preparation is technically pure (homogeneous), but that it is pure enough to provide the polypeptide or nucleic acid in a form that can be used for the intended purpose.

[0056] The term "fragment" applied to a polypeptide will be understood to refer to an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence, and comprising, consisting essentially of, and / or consisting of, a continuous amino acid sequence of amino acids that is consistent or almost consistent (e.g., 90%, 92%, 95%, 98%, 99% consistent) with the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the present invention may be included in a larger polypeptide as a component under appropriate circumstances. In some embodiments, such a fragment may include, consist essentially of, and / or consist of, a peptide having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more continuous amino acids of a polypeptide or amino acid sequence according to the present invention.

[0057] "Vector" is any nucleic acid molecule used to clone and / or transfer nucleic acid into a cell. A vector can be a replicon to which another nucleotide sequence can be attached to allow the attached nucleotide sequence to replicate. "Replicon" can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) as an autonomous unit of nucleic acid replication in vivo (i.e., capable of replicating under its own control). The term "vector" includes viral and non-viral (e.g., plasmid) nucleic acid molecules that introduce nucleic acid into cells in vitro, in vitro, and / or in vivo. Nucleic acids can be manipulated using a large number of vectors known in the art, responsive elements and promoters can be merged into genes, etc. For example, a nucleic acid fragment corresponding to a response element and a promoter can be inserted into a suitable vector, which can be achieved by connecting a suitable nucleic acid fragment to a selected vector with complementary binding ends. Alternatively, the ends of nucleic acid molecules can be enzymatically modified, or any site can be produced by connecting a nucleotide sequence (joint) to a nucleic acid end. Such a vector can be engineered to contain a sequence encoding a selective marker, which facilitates the selection of cells containing the vector and / or cells having the nucleic acid of the vector merged into the cell genome. Such markers allow identification and / or selection of host cells that incorporate and express the protein encoded by the marker.A "recombinant" vector refers to a viral or non-viral vector comprising one or more heterologous nucleotide sequences (i.e., a transgene), for example comprising two, three, four, five or more heterologous nucleotide sequences.

[0058] Viral vectors have been used in various gene delivery applications in cells and living animal subjects. Available viral vectors include but are not limited to retrovirus, slow virus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus and adenovirus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cytofectin), nucleic acid-protein complexes and biopolymers. In addition to the nucleic acid of interest, the vector can also include one or more regulatory regions and / or can be used for selecting, measuring and monitoring nucleic acid transfer results (delivery to specific tissues, expression duration, etc.) selectable markers.

[0059] The vector can be introduced into the desired cells by methods known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun or a nucleic acid carrier transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al. Canadian Patent Application No. 2,012,311 filed March 15, 1990). In various embodiments, other molecules can be used to facilitate delivery of nucleic acids in vivo, such as cationic oligopeptides (e.g., WO95 / 21931), peptides derived from nucleic acid binding proteins (e.g., WO96 / 25508) and / or cationic polymers (e.g., WO95 / 21931). Vectors can also be introduced in vivo as naked nucleic acids (see U.S. Pat. Nos. 5,693,622, 5,589,466, and 5,580,859). Receptor-mediated nucleic acid delivery methods can also be used (Curiel et al., Hum. Gene Ther. 3:147 (1992); Wu et al., J. Biol. Chem. 262:4429 (1987)).

[0060] As used herein, the terms "protein" and "polypeptide" are used interchangeably and include both peptides and proteins unless otherwise indicated.

[0061] "Fusion protein" is a polypeptide produced when two heterologous nucleotide sequences encoding two (or more) different polypeptides, or fragments thereof, which are not found fused together in nature, are fused together in the correct translation reading frame. Illustrative fusion polypeptides include fusions of the polypeptide of the present invention (or fragments thereof) with all or part of glutathione-S-transferase, maltose binding protein or reporter protein (e.g., green fluorescent protein, β-glucuronidase, β-galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, etc.

[0062] As used herein, a "functional" polypeptide or "functional fragment" is a substance that substantially retains at least one biological activity normally associated with the polypeptide (e.g., angiogenic activity, protein binding, ligand or receptor binding). In a specific embodiment, a "functional" polypeptide or "functional fragment" substantially retains all activities possessed by an unmodified peptide. By "substantially retaining" biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 99% or more of the biological activity of the native polypeptide (and may even have a higher level of activity than the native polypeptide). A "non-functional" polypeptide is a polypeptide that exhibits little or substantially no detectable biological activity normally associated with a polypeptide (e.g., at most an insignificant amount, such as less than about 10% or even 5%). Biological activities such as protein binding and angiogenic activity can be measured using assays well known in the art and as described herein.

[0063] The term "express" or "expressed" a polynucleotide coding sequence means that the sequence is transcribed and optionally translated. Generally, according to the present invention, the expression of the coding sequence of the present invention will result in the production of the polypeptide of the present invention. The entire expressed polypeptide or fragment can also function in intact cells without purification.

[0064] In the context of the present invention, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV, as well as any other AAV now known or later discovered. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th Edition, Lippincott-Raven Publishers). Several additional AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virol. 78:6381-6388 and Table 1), which are also encompassed by the term "AAV".

[0065] The genome sequences of various AAVs and autonomous parvoviruses, as well as the sequences of ITRs, Rep proteins, and capsid subunits are known in the art. These sequences can be found in the literature or in public databases, such as Database. See e.g. Accession No. NC 002077, NC 001401, NC 001729, NC 001863, NC 001829, NC001862, NC 000883, NC 001701, NC 001510, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC001358, NC 001540, AF513851, AF513852, AY530579, AY631965, AY631966; the disclosures of which are incorporated herein in their entirety. See also, e.g., Srivistava et al., (1983) J. Virol. 45:555; Chiorini et al., (1998) J. Virol. 71:6823; Chiorini et al., (1999) J. Virol. 73:1309; Bantel-Schaal et al., (1999) J. Virol. 73:939; Xiao et al., (1999) J. Virol. 73:3994; Muramatsu et al., (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854; International Patent Publication Nos. WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; U.S. Pat. No. 6,156,303; the disclosures of which are incorporated herein in their entirety. See also Table 1. Xiao, X., (1996), "Characterization of Adeno-associated virus (AAV) DNA replication and integration," Ph.D. Dissertation, University of Pittsburgh, Pittsburgh, PA (incorporated herein in its entirety) provides an early description of the terminal repeat sequences of AAV1, AAV2, and AAV3.

[0066] Table 1

[0067]

[0068]

[0069] A "recombinant AAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) comprising at least one inverted terminal repeat (e.g., one, two, or three inverted terminal repeat sequences) and one or more heterologous nucleotide sequences. The rAAV vector typically retains 145 base terminal repeats (TRs) in cis to produce the virus; however, modified AAV TRs and non-AAV TRs (including partially or completely synthetic sequences) can also be used for this purpose. All other viral sequences are unnecessary and can be provided in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158: 97). The rAAV vector optionally comprises two TRs (e.g., AAV TRs), which will typically be at the 5' and 3' ends of the heterologous nucleotide sequence, but not necessarily adjacent to it. TRs may be identical to or different from each other. The vector genome may also contain a single ITR at its 3' or 5' end.

[0070] The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates a desired function such as replication, viral packaging, integration and / or proviral rescue, etc.). TRs may be AAV TRs or non-AAV TRs. For example, non-AAV TR sequences, such as those of other parvoviruses (e.g., canine parvovirus (CPV), parvovirus of mice (MVM), human parvovirus B-19), or sequences of the SV40 hairpin that serve as the SV40 origin of replication, may be used as TRs, which may be further modified by truncation, substitution, deletion, insertion and / or addition. In addition, TRs may be partially or completely synthetic, such as the "double D sequence" described in U.S. Pat. No. 5,478,745 to Samulski et al.

[0071] "AAV terminal repeats" or "AAV TRs" can be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV now known or later discovered (see, e.g., Table 1). The AAV terminal repeats need not have native terminal repeat sequences (e.g., native AAV TR sequences may be altered by insertions, deletions, truncations, and / or missense mutations), as long as the terminal repeats mediate the desired function, e.g., replication, viral packaging, integration, and / or proviral rescue, etc.

[0072] The terms "rAAV particle" and "rAAV viral particle" are used interchangeably herein. An "rAAV particle" or "rAAV viral particle" comprises a rAAV vector genome packaged within an AAV capsid.

[0073] AAV capsid structure is described in more detail in BERNARD N. FIELDS et al., VIROLOGY, Vol. 2, Chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).

[0074] The term "pharmacokinetic properties" has its usual and customary meaning and refers to the absorption, distribution, metabolism and excretion of the FVIII protein.

[0075] The usual and customary meaning of "bioavailability" is the fraction or amount of an administered dose of a biologically active drug that reaches the systemic circulation. In the context of embodiments of the present invention, the term "bioavailability" includes the usual and customary meaning, but is also considered to have a broader meaning to include the extent of biological activity of a FVIII protein. In the case of FVIII, for example, one measure of "bioavailability" is the procoagulant activity of the FVIII protein available in the circulation after infusion.

[0076] "Post-translational modification" has the usual and customary meaning and includes, but is not limited to, removal of leader sequences, γ-carboxylation of glutamic acid residues, β-hydroxylation of aspartic acid residues, N-linked glycosylation of asparagine residues, O-linked glycosylation of serine and / or threonine residues, sulfation of tyrosine residues, phosphorylation of serine residues, and any combination thereof.

[0077] As used herein, "biological activity" is determined by reference to a standard, e.g., from human plasma. For FVIII, the standard may be (CSL Behring). The biological activity of this standard was taken as 100%.

[0078] The term "factor VIII protein" or "FVIII protein" as used herein includes wild-type FVIII protein and naturally occurring or artificial proteins (e.g., proteins with B domain deletions). The FVIII protein of the present invention may further include mutant forms of FVIII known in the literature. The FVIII protein of the present invention also includes any other naturally occurring human FVIII protein or artificial human FVIII protein now known or later identified, as well as their derivatives and active fragments / active domains known in the art.

[0079] The amino acid sequences of FVIII from various mammalian species are available from sequence databases such as GenBank. Examples of FVIII sequences are shown in the table below.

[0080] Species GenBank Accession Number Homo sapiens AAA52484.1 House mouse (Mus musculus) NP_032003.2 Wild boar (Sus scrofa) AAB06705.1 Bos Taurus NP_001138980.1 Domestic dog (Canis lupus familiaris) NP-001003212.1 Brown rat (Rattus norvegicus) ADU79112.1

[0081] The FVIII protein of the present invention also includes a pharmacologically active form of FVIII, which is a molecule from which the signal peptide has been removed, and the B domain has been removed by the action of a protease (or engineered from the protein by removing it at the nucleic acid level), resulting in two discontinuous polypeptide chains (light chain and heavy chain) of FVIII folded into a functional FVIII coagulation factor. Human FVIII is known to have multiple B domain-deleted forms, including the frequently used SQ version, in which the residues between S743 and Q1638 are deleted. Specifically, modified FVIII proteins with increased glycosylation are specifically included in a broad sense.

[0082] The amino acid sequence of human FVIII protein is well known in the art and can be found in GenBank accession number AAA52484. The length of human FVIII protein is 2351 amino acids and consists of a signal peptide (residues 1-19), a heavy chain (residues 20-759), a B domain (residues 760-1332) and a light chain (residues 1668-2351). The amino acid sequence without a signal peptide is disclosed below (SEQ ID NO: 5).

[0083] The term "half-life" is a broad term that includes the usual and customary meanings and the usual and customary meanings found in the FVIII scientific literature. The definition specifically includes the measurement of parameters related to FVIII, which defines the time after the infusion from the initial value measured during infusion to the half of the initial value. In some embodiments, the half-life of FVIII can be measured in blood and / or blood components using antibodies to FVIII in various immunoassays, as well known in the art and as described herein. Alternatively, functional assays including standard coagulation assays can be used to measure half-life with a reduction in FVIII activity, as well known in the art and as described herein.

[0084] The term "recovery" as used herein includes, after its infusion, injection, delivery or administration in other ways, in order to measure the level of FVIII, the amount of FVIII measured by any acceptable method in a recipient animal or human subject (e.g., in the circulation) at the earliest practical time of taking out a biological sample (e.g., a blood or blood product sample), including but not limited to the level of FVIII antigen detected or the level of FVIII protease or coagulation activity. Using existing methods, the earliest biological sampling time for measuring FVIII recovery is usually within the first 15 minutes after the infusion, injection or other delivery / administration of FVIII, but with the improvement of science and / or clinical technology, it is reasonable to expect faster sampling time. In essence, the recovery value of FVIII is represented here, at the earliest possible time point after infusion, injection or other delivery to a recipient animal or patient, the maximum fraction of FVIII infused, injected or otherwise delivered / administered that can be measured in a recipient (e.g., in the circulation).

[0085] The term "glycosylation site" is a broad term with its usual and customary meaning. In the context of the present application, the term applies to both sites that can potentially accept carbohydrate moieties as well as sites within a protein, particularly FVIII, to which carbohydrate moieties have actually been attached and includes any amino acid sequence that can serve as an acceptor for oligosaccharides and / or carbohydrates.

[0086] As used herein, a "transformed" cell is a cell that has been transformed, transduced and / or transfected with a nucleic acid molecule encoding a FVIII protein of the present invention, including but not limited to a FVIII protein vector constructed using recombinant DNA technology.

[0087] As used herein, the term "bleeding disorder" reflects any defect of cellular, physiological or molecular origin, congenital, acquired or induced, in which bleeding is manifested. Examples are coagulation factor deficiencies (e.g., hemophilia A and B or deficiencies of coagulation factors XI, VII, VIII or IX), coagulation factor inhibitors, platelet insufficiency, thrombocytopenia, von Willebrand's disease, or bleeding caused by surgery or trauma.

[0088] Excessive bleeding also occurs in subjects with a normally functioning blood coagulation cascade (without coagulation factor deficiency or inhibitors for any coagulation factor), and may be caused by platelet insufficiency, thrombocytopenia, or von Willebrand disease. In this case, bleeding may be similar to bleeding caused by hemophilia, because the hemostatic system (as in hemophilia) lacks or has abnormal necessary coagulation "compounds" (such as platelets or von Willebrand factor proteins), resulting in major bleeding. In subjects experiencing extensive tissue damage associated with surgery or trauma, normal hemostatic mechanisms may be overwhelmed by the need for immediate hemostasis, so bleeding may still occur despite normal hemostatic mechanisms. When bleeding in organs such as the brain, inner ear region, and eyes, the possibility of surgical hemostasis is limited, and achieving satisfactory hemostasis is also a problem. The same problem may also occur during biopsy in various organs (liver, lung, tumor tissue, gastrointestinal tract) and laparoscopic surgery. All these cases have in common that it is difficult to provide hemostasis by surgical techniques (sutures, clips, etc.), also when the bleeding is diffuse (hemorrhagic gastritis and heavy uterine bleeding). Acute and heavy bleeding may also occur in subjects treated with anticoagulation, in which defective hemostasis is induced by the treatment given. In the case where the anticoagulation must be quickly offset, these subjects may need surgical intervention. Radical retropubic prostatectomy is a conventional operation for subjects with localized prostate cancer. The operation is often complicated by significant and sometimes massive blood loss. Considerable blood loss during prostatectomy is mainly related to complex anatomical conditions, which have various densely vascularized parts that are not easy to obtain surgical hemostasis, and may cause large areas of diffuse bleeding. In addition, intracerebral hemorrhage is the most untreatable form of stroke and is related to high mortality and hematoma growth in the first few hours after intracerebral hemorrhage. Another situation that may cause problems in the case of poor hemostasis is when subjects with normal hemostatic mechanisms are treated with anticoagulation to prevent thromboembolic diseases. Such treatments may include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K antagonists, and aspirin and other platelet aggregation inhibitors.

[0089] In one embodiment of the invention, the bleeding is associated with hemophilia. In another embodiment, the bleeding is associated with hemophilia with acquired inhibitors. In another embodiment, the bleeding is associated with thrombocytopenia. In another embodiment, the bleeding is associated with von Willebrand's disease. In another embodiment, the bleeding is associated with severe tissue damage. In another embodiment, the bleeding is associated with severe trauma. In another embodiment, the bleeding is associated with surgery. In another embodiment, the bleeding is associated with laparoscopic surgery. In another embodiment, the bleeding is associated with hemorrhagic gastritis. In another embodiment, the bleeding is a large amount of uterine bleeding. In another embodiment, the bleeding occurs in an organ with limited mechanical hemostasis possibilities. In another embodiment, the bleeding occurs in the brain, the inner ear region, or the eye. In another embodiment, the bleeding is associated with the process of taking a biopsy. In another embodiment, the bleeding is associated with anticoagulant therapy.

[0090] "Subjects" of the present invention include any animal suffering from or susceptible to bleeding disorders or bleeding conditions, which require and / or expect to control bleeding, which can be treated, improved or prevented by administering FVIII to the subject (e.g., hemophilia A and acquired FVIII deficiency (e.g., due to autoantibodies to FVIII or hematological malignancies)). Such subjects are typically mammalian subjects (e.g., laboratory animals such as rats, mice, guinea pigs, rabbits, primates, etc.), farm or commercial animals (e.g., cows, horses, goats, donkeys, sheep, etc.), or livestock (e.g., cats, dogs, ferrets, etc.). In a specific embodiment, the subject is a primate subject, a non-human primate subject (e.g., chimpanzee, baboon, monkey, gorilla, etc.) or a human being. The subject of the present invention may be a subject known or believed to have a bleeding disorder or bleeding condition risk that needs and / or expects to be controlled. Alternatively, the subject according to the present invention may also include a subject previously unknown or suspected of having a bleeding disorder or bleeding condition risk that needs or expects to be controlled. As another option, the subject may be a laboratory animal and / or an animal model of the disease.

[0091] Subjects include males and / or females of any age, including newborns, infants, adults and elderly subjects. With regard to human subjects, in representative embodiments, subjects can be infants (e.g., less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months or less age), toddlers (e.g., at least about 12, 18 or 24 months and / or less than about 36, 30 or 24 months) or children (e.g., at least about 1, 2, 3, 4 or 5 years old and / or less than about 14 years old, 12, 10, 8, 7, 6, 5 or 4 years old). In embodiments of the invention, the subject is a human subject of about 0 to 3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 3 to 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 6 to 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 9 to 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 12 to 18, 24, 36, 48 or 60 months of age, a human subject of about 18 to 24, 30, 36, 48 or 60 months of age, or a human subject of about 24 to 30, 36, 48 or 60 months of age.

[0092] Promoter and expression cassette

[0093] One aspect of the present invention relates to a polynucleotide comprising a synthetic liver-specific promoter, wherein the promoter comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1 or a sequence having at least about 90% identity thereto. In some embodiments, the nucleotide sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of SEQ ID NO: 1. The promoter is a short (about 200 base pairs) and strong liver-specific promoter, which is ideal for liver-specific expression of a polynucleotide of interest and is particularly suitable for use in AAV vectors due to its short length and the limited capacity of AAV vectors. The promoter is designed to contain a conserved basic promoter element and a transcription start site. The basic promoter is connected at its 5' end to a number of liver-specific transcription factor binding sites for liver-specific expression ( Figure 1 The promoter was initially identified in vitro using a luciferase reporter gene and transfection experiments in a human hepatoma cell line Huh7, and then confirmed in vivo in mice to show high activity.

[0094] In some embodiments, the promoter is part of an expression cassette, wherein it is operably connected to an intron, such as on the 3' end of the promoter. This can be done to increase the expression level of the polynucleotide of interest connected to the promoter. Any suitable intron can be used, such as the chimeric intron CIN (Promega). In some embodiments, the intron is from VH4. In some embodiments, the intron can further include a short non-natural exon junction sequence. In one embodiment, the promoter and intron together include the nucleotide sequence of SEQ ID NO:2 or a sequence having at least 90% identity therewith (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence of SEQ ID NO:1), or consist essentially of it, or consist of it.

[0095] The promoter may be operably linked to a polynucleotide of interest. In some embodiments, the polynucleotide of interest encodes a polypeptide or a functional nucleic acid. In certain embodiments, the polynucleotide of interest encodes a coagulation factor, such as FVIII, such as FVIII with a B domain deletion. The FVIII with a B domain deletion may be encoded by a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 3 or a sequence having at least 90% identity therewith (e.g., with a nucleotide sequence of SEQ ID NO: 3 having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity), or consisting essentially of, or consisting of. In one embodiment, the expression cassette comprising a promoter, an intron, and a polynucleotide of interest encoding a B-domain deleted FVIII comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 4, or a sequence having at least 90% identity thereto (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of SEQ ID NO: 4).

[0096] Another aspect of the present invention is a vector comprising a polynucleotide of the present invention, such as an expression vector. The vector can be any type of vector known in the art, including but not limited to plasmid vectors and viral vectors. In some embodiments, the viral vector is a retroviral or lentiviral vector. In some embodiments, the viral vector is an AAV vector from any known AAV serotype, including but not limited to AAV type 1, AAV type 2, AAV type 3 (including 3A type and 3B type), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV11, bird AAV, cattle AAV, dog AAV, horse AAV and sheep AAV and any other AAV known now or later found. In some embodiments, the AAV vector is AAV8 or AAV9.

[0097] Another aspect of the invention relates to cells (e.g., isolated cells, transformed cells, recombinant cells, etc.) comprising a polynucleotide and / or vector of the invention. Thus, various embodiments of the invention relate to recombinant host cells containing a vector (e.g., an expression cassette). Such cells may be isolated and / or present in a transgenic animal. Transformation of cells is further described below.

[0098] Another aspect of the present invention relates to a transgenic animal comprising a polynucleotide, vector and / or transformed cell of the present invention. Transgenic animals are further described below.

[0099] The polynucleotides, vectors and / or cells of the invention may be included in pharmaceutical compositions. Some embodiments relate to kits comprising the polynucleotides, vectors and / or cells of the invention, and / or reagents and / or instructions for using the kits, for example, to implement the methods of the invention.

[0100] Modified Factor VIII Protein

[0101] One aspect of the present invention relates to modified mammalian factor VIII polypeptides (e.g., human FVIII polypeptides), wherein the amino acid residues in the heavy chain are modified to produce one or more additional glycosylation sites. In certain embodiments, the one or more additional glycosylation sites are located in the C-terminus of the heavy chain, such as the last 100 amino acid residues of the heavy chain, such as the last 50, 40, 30, 20, 10, 9, 8, 7, 6 or 5 residues. In some embodiments, the polypeptide is modified to produce at least 2 glycosylation sites, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more glycosylation sites. Modifications may include amino acid substitutions, additions, deletions or any combination thereof. These modifications are introduced into the amino acid sequence of the FVIII protein to produce a FVIII protein with increased activity after expression in vivo.

[0102] "Additional" glycosylation sites refers to the number of glycosylation sites in the FVIII protein being greater than the number of glycosylation sites that are normally present in an unmodified (eg, wild-type) FVIII protein (eg, SEQ ID NO: 5).

[0103] The present invention also relates to a FVIII protein containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) additional sugar side chains. Such additional sugar side chains may be present at one or more glycosylation sites in the FVIII protein of the present invention. Alternatively, as is well known to those skilled in the art, the additional sugar side chains may be present at sites on the FVIII protein as a result of chemical and / or enzymatic methods of introducing such sugar chains into the FVIII molecule. "Additional" or "new" sugar chains refer to the number of sugar chains in the FVIII protein that is greater than the number of sugar chains that are normally present in the "wild type" form of FVIII. In various embodiments, from about 1 to about 50 additional sugar side chains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) may be added.

[0104] The glycosylation site can be an N-linked glycosylation site, an O-linked glycosylation site, and a combination of an N-linked glycosylation site and an O-linked glycosylation site. In some embodiments, the added glycosylation site includes an N-linked glycosylation site, and the consensus sequence is NXT / S, provided that X is not proline. In other embodiments, the glycosylation site comprises an O-linked glycosylation site containing a consensus sequence selected from the group consisting of CXXGGT / SC (SEQ ID NO: 24), NSTE / DA (SEQ ID NO: 25), NITQS (SEQ ID NO: 26), QSTQS (SEQ ID NO: 27), D / E-FT-R / KV (SEQ ID NO: 28), CE / D-SN (SEQ ID NO: 29), GGSC-K / R (SEQ ID NO: 30), and any combination thereof.

[0105] In some embodiments, about 1 to about 15 glycosylation sites can be added to the amino acid sequence of the FVIII protein of the invention. In various embodiments, about 1 to about 50 glycosylation sites (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) can be added.

[0106] As used herein, "glycosylation attachment site" or "glycosylation site" may refer to a sugar attachment consensus sequence (i.e., a series of amino acids that serve as a consensus sequence for attaching a sugar (monosaccharide, oligosaccharide, or polysaccharide) to an amino acid sequence) or it may refer to the actual amino acid residue to which the sugar moiety is covalently linked. The sugar moiety may be a monosaccharide (simple sugar molecule), an oligosaccharide, or a polysaccharide.

[0107] In a specific embodiment, additional amino acids may be inserted between any amino acid residues constituting the heavy chain and / or substituted into such amino acid residues. In addition, the same insert of the present invention may be introduced multiple times at the same and / or different positions of the amino acid sequence of the FVIII protein. In addition, different inserts and / or the same insert may be introduced once or multiple times at the same and / or different positions between the amino acid residues of the entire amino acid sequence of the FVIII protein.

[0108] Some proteins can support a large number of sugar side chains, and the distance between N-linked glycosylation sites can be as few as three, four, five, or six amino acids (see, e.g., Lundin et al., FEBS Lett. 581:5601 (2007); Apweiler et al., Biochim. Biophys. Acta 1473:4 (1991), the entire contents of which are incorporated herein by reference).

[0109] In some embodiments, amino acid residues 736 and 737 of the wild-type human sequence (SEQ ID NO: 5) are replaced with amino acid residue XX, wherein X is S or T. Thus, residues 736 and 737 may be SS, ST, TS, or TT.

[0110] In some embodiments, amino acid residues 736-742 of the wild-type human sequence (SEQ ID NO: 5) are replaced with amino acid residues XXYVNRXL (SEQ ID NO: 6), wherein X is S or T. Thus, residues 736-742 may be as follows.

[0111] Residues 736-742 SEQ ID NO TTYVNRSL 7 TTYVNRTL 8 TSYV 9 TSV 10 STYVNRSL 11 STYVNRTL 12 SSYVNRSL 13 SSYVNRTL 14

[0112] In some embodiments, amino acid residues 736-742 in the wild-type human sequence (SEQ ID NO: 5) are replaced with amino acid residues XXNNX (SEQ ID NO: 15), wherein X is S or T. Thus, residues 736-740 may be as follows.

[0113]

[0114]

[0115] In some embodiments, the modified human Factor VIII polypeptide is one in which the B domain is deleted, such as an SQ deletion from S743 to Gln1638 (as numbered in SEQ ID NO: 5).

[0116] The FVIII protein of the present invention having additional glycosylation sites can be produced by recombinant methods such as site-directed mutagenesis using PCR. Alternatively, the FVIII protein of the present invention can be chemically synthesized to prepare a FVIII protein having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) additional glycosylation sites.

[0117] It is within the skill of those skilled in the art and within the scope of the present invention to modify any amino acid residue or residues in the mature FVIII amino acid sequence according to methods well known in the art and as taught herein, and to test any resulting FVIII protein for activity, stability, recovery, half-life, etc. according to methods well known in the art and as taught herein (see, e.g., Elliott et al., J. Biol. Chem. 279: 16854 (2004), the entire contents of which are incorporated herein by reference).

[0118] Embodiments of the present invention relate to recombinant FVIII proteins (e.g., X0, X1, X2), wherein glycosylation sites have been added to improve the activity and / or recovery and / or half-life and / or stability of FVIII. The FVIII protein of the present invention comprises a modification that allows the bioavailability of the FVIII protein to be increased for a subject to which the FVIII protein of the present invention has been administered. In some embodiments, the improved bioavailability refers to the standard current thinking in hematology, and the concentration of FVIII in plasma is the relevant concentration. In some embodiments of the present invention, the improved bioavailability refers to the ability of the FVIII protein to stay longer in the circulation of the subject. Therefore, in some embodiments of the present invention, the FVIII protein described herein is modified to produce a FVIII protein with increased activity after in vivo expression, and in some embodiments, the present invention provides a method for increasing the hemostatic effectiveness of the FVIII protein in a subject, comprising administering an effective amount of the FVIII protein of the present invention, the polynucleotide of the present invention, the vector of the present invention, and / or the cell of the present invention to the subject, wherein the FVIII protein administered to the subject in any of these embodiments is a FVIII protein of the present invention with increased activity.

[0119] FVIII protein according to the present invention is produced and characterized by methods well known in the art and described herein. As well known in the art, these methods include measuring clotting time (partial thromboplastin time (PPT) assay)) and administering FVIII protein to test animals to determine recovery, half-life and bioavailability by appropriate immunoassays and / or activity assays.

[0120] Another aspect of the present invention provides an isolated polynucleotide encoding the FVIII protein of the present invention and an expression cassette for producing the FVIII protein.

[0121] Another aspect of the present invention is a vector comprising a polynucleotide of the present invention, such as an expression vector. The vector can be any type of vector known in the art, including but not limited to plasmid vectors and viral vectors. In some embodiments, the viral vector is an AAV vector from any known AAV serotype, including but not limited to this AAV type 1, AAV type 2, AAV type 3 (including type 3A and type 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV11, bird AAV, cattle AAV, dog AAV, horse AAV and sheep AAV, and any other AAV now known or later discovered. In some embodiments, the AAV vector is AAV8 or AAV9.

[0122] Another aspect of the invention relates to cells (e.g., isolated cells, transformed cells, recombinant cells, etc.) comprising the polynucleotides and / or vectors of the invention. Thus, various embodiments of the invention relate to recombinant host cells containing the vectors (e.g., expression cassettes). Such cells may be isolated and / or present in transgenic animals. Transformation of cells is further described below.

[0123] Another aspect of the present invention relates to a transgenic animal comprising a polynucleotide, vector and / or transformed cell of the present invention. Transgenic animals are further described below.

[0124] The FVIII proteins, polynucleotides, vectors and / or cells of the invention may be included in pharmaceutical compositions. Some embodiments relate to kits comprising the FVIII proteins, polynucleotides, vectors and / or cells of the invention, and / or reagents and / or instructions for using the kits, e.g., to practice the methods of the invention.

[0125] Method of the present invention

[0126] Another aspect of the present invention relates to promoter of the present invention and expression cassette for producing the purposes of polypeptide or functional nucleic acid in a liver-specific manner, for example.Therefore, one aspect relates to the method for producing polypeptide or functional nucleic acid in the liver of experimenter, comprises sending polynucleotide of the present invention, carrier and / or transformed cell to experimenter, thereby produces polypeptide or functional nucleic acid acid in the liver of experimenter.Described polynucleotide, carrier and / or transformed cell are sent under the condition that interested polynucleotide expression occurs, to produce polypeptide or functional nucleic acid.These conditions are well-known in the art and are described further below.

[0127] Another aspect of the invention relates to a method of treating hemophilia A or acquired factor VIII deficiency in a subject using the promoter and expression cassette of the invention, comprising delivering to the subject a therapeutically effective amount of a polynucleotide, vector and / or transformed cell of the invention, thereby treating hemophilia A in the subject. In some embodiments, the polynucleotide of interest encodes a FVIII polypeptide as described above.

[0128] Another aspect of the invention relates to a method for increasing the bioavailability of a FVIII polypeptide in a subject using the promoter and expression cassette of the invention, comprising delivering an effective amount of a polynucleotide, vector and / or transformed cell of the invention to the subject, thereby increasing the bioavailability of the FVIII polypeptide in the subject. In this aspect, the polynucleotide of interest encodes a FVIII polypeptide as described above.

[0129] The modified FVIII proteins of the present invention can be used in methods for treating bleeding disorders by administering an effective amount of the FVIII protein to a subject (e.g., a human patient) in need thereof. Thus, the present invention also provides methods for treating bleeding disorders, comprising administering an effective amount of the FVIII protein, polynucleotide, vector and / or cell of the present invention to a subject in need thereof.

[0130] One aspect of the present invention relates to a method for producing Factor VIII in the liver of a subject, comprising delivering to the subject a polynucleotide, vector and / or transformed cell encoding a modified human Factor VIII polypeptide of the present invention, thereby producing Factor VIII in the liver of the subject.

[0131] Another aspect of the invention relates to a method of treating hemophilia A or acquired Factor VIII deficiency in a subject, comprising delivering to the subject a therapeutically effective amount of a modified human Factor VIII polypeptide, polynucleotide, vector and / or transformed cell of the invention, thereby treating hemophilia A or acquired Factor VIII deficiency in the subject.

[0132] Another aspect of the present invention relates to a method for increasing the bioavailability of a Factor VIII polypeptide in a subject, comprising delivering to the subject an effective amount of a polynucleotide, vector and / or transformed cell encoding a modified human Factor VIII polypeptide of the present invention, thereby increasing the bioavailability of the Factor VIII polypeptide in the subject.

[0133] Bleeding disorders that can be treated according to the methods of the invention include any disorder that can be treated with FVIII, such as hemophilia A and acquired FVIII deficiency. Such treatment protocols and dosing regimens for administering or delivering a FVIII protein of the invention and / or a polynucleotide encoding a FVIII protein of the invention to a subject (e.g., a subject in need thereof) are well known in the art.

[0134] In an embodiment of the present invention, the dosage of a vector (e.g., a viral vector or other nucleic acid vector) encoding a FVIII protein of the present invention can be an amount that achieves a therapeutic plasma concentration of the FVIII protein. The therapeutic concentration of the FVIII protein is considered to be higher than the normal level of 1% of healthy individuals, which is measured on an average of 100%, thus being one international unit (IU) of FVIII in 1mL of normal human plasma. Those skilled in the art will be able to determine the optimal dosage for a given subject and a given condition.

[0135] For treatment associated with intentional intervention, the FVIII protein of the invention is typically administered within about 24 hours prior to the intervention and for up to 7 days or more thereafter.Administration as a coagulant can be by a variety of routes as described herein.

[0136] The pharmaceutical composition is mainly used for parenteral administration for preventive and / or therapeutic treatment. Preferably, the pharmaceutical composition is parenteral, i.e., intravenous, subcutaneous or intramuscular administration, or administered by continuous or pulse infusion. Alternatively, the pharmaceutical composition can be formulated for administration in various ways, including but not limited to oral, subcutaneous, intravenous, intracerebral, intranasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, intraocular or any other acceptable way.

[0137] Compositions for parenteral administration include the FVIII protein of the present invention in combination with (e.g., dissolved in) a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine, etc. Compositions that extend stability and storage (such as methionine and sucrose) can also be used to formulate the FVIII protein of the present invention. The FVIII protein of the present invention can also be formulated into liposome preparations for delivery or targeting to the site of injury. Liposomal preparations are generally described in U.S. Patent Nos. 4,837,028, 4,501,728, and 4,975,282. The composition can be sterilized by conventional well-known sterilization techniques. The resulting aqueous solution can be packaged for use, or filtered and lyophilized under sterile conditions, and the lyophilized preparation is combined with a sterile aqueous solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances required for approximate physiological conditions, such as pH adjustment and buffers, tension regulators, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. The composition may further contain a preservative, an isotonic agent, a nonionic surfactant or detergent, an antioxidant and / or other various additives.

[0138] The concentration of FVIII protein in these formulations can vary widely, i.e., from less than about 0.5% by weight (typically at or at least about 1% by weight) to as much as about 15% or 20% by weight, and will be selected primarily based on the particular mode of administration selected by fluid volume, viscosity, etc. Thus, as a non-limiting example, a typical pharmaceutical composition for intravenous infusion can be prepared to contain 250 ml of sterile Ringer's solution and 10 mg of FVIII protein. Actual methods for preparing parenterally administrable compositions are known or apparent to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences A more detailed description is given in , 21st ed., Mack Publishing Company, Easton, Pa. (2005).

[0139] Compositions comprising the FVIII protein of the present invention and / or nucleic acid molecules encoding the FVIII protein of the present invention can be administered for preventive and / or therapeutic treatments. In therapeutic applications, the composition is administered to a subject already suffering from the disease in an amount sufficient to cure, alleviate or partially prevent the disease and its complications as described above. An amount sufficient to achieve this goal is defined as a "therapeutically effective amount". As will be appreciated by those skilled in the art, the amount effective for this purpose will depend on the severity of the disease or injury and the weight and general state of the subject.

[0140] In prophylactic applications, a composition containing a FVIII polypeptide of the invention is administered to a subject susceptible to or otherwise at risk of a disease state or injury to enhance the subject's own coagulation ability. Such an amount is defined as a "prophylactically effective dose". In prophylactic applications, the exact amount again depends on the subject's health and weight.

[0141] Single or multiple administrations of the composition can be carried out at a dose level and pattern selected by the treating physician.For ambulatory subjects requiring daily maintenance levels, the FVIII protein can be administered by continuous infusion using, for example, an ambulatory pump system.

[0142] The FVIII proteins of the present invention may also be formulated as sustained or extended release formulations. Methods for formulating sustained or extended release compositions are known in the art and include, but are not limited to, semipermeable matrices of solid hydrophobic particles containing the polypeptide.

[0143] Local delivery of the FVIII protein of the present invention, such as local administration, can be performed, for example, by spraying, perfusion, double balloon catheters, stents, incorporation into vascular grafts or stents, hydrogels for coating balloon catheters, or other established methods. In any case, the pharmaceutical composition should provide an amount of FVIII protein sufficient to effectively treat the subject.

[0144] In some embodiments, an AAV vector is used to deliver a polynucleotide of interest (e.g., a FVIII protein) to a subject. Accordingly, the present invention also provides an AAV viral particle (i.e., a virion) comprising a polynucleotide of interest, wherein the viral particle packages (i.e., encapsidates) a vector genome, optionally an AAV vector genome.

[0145] In a specific embodiment, a virion is a recombinant vector comprising a heterologous polynucleotide of interest (e.g., for delivery to a cell). Therefore, the present invention can be used to deliver a polynucleotide to a cell in vitro, in vitro, and in vivo. In a representative embodiment, a recombinant vector of the present invention can be advantageously used to deliver or transfer a polynucleotide to an animal (e.g., mammal) cell.

[0146] Any heterologous nucleotide sequence may be delivered by the viral vectors of the invention.Polynucleotides of interest include those encoding polypeptides, optionally therapeutic (eg, for medical or veterinary use) and / or immunogenic (eg, vaccine) polypeptides.

[0147] Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulator (CFTR), dystrophin (including protein products of dystrophin mini-genes or micro-genes, see, e.g., Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent Application No. 2003017131; Wang et al., (2000) Proc. Natl. Acad. Sci. USA 97:13714-9 [min-dystrophin]; Harper et al., (2002) Nature Genetics 5 ... Med.8:253-61 [micro-dystrophin]); min-agrin, laminin-α2, sarcoglycan (α, β, γ or δ), Fukutin-related protein, myostatin propeptide, follistatin, dominant negative myostatin, angiogenic factors (e.g., VEGF, angiopoietin-1 or 2), anti-apoptotic factors (e.g., heme oxygenase-1, TGF-β, inhibitors of pro-apoptotic signaling such as caspases, proteases, kinases, death receptors [e.g., CD-095], cytochrome c release regulators, inhibitors of mitochondrial pore opening and swelling); activin type II soluble receptor, anti-inflammatory polypeptides such as Ikappa B dominant mutants, sarcospan, utrophin, mini-utrophin, antibodies or antibody fragments directed against myostatin or myostatin propeptide, cell cycle regulators, Rho kinase regulators such as Cethrin, which is a modified bacterial C3 exoenzyme [available from BioAxone Therapeutics, Inc., Saint-Lauren, Quebec, Canada], BCL-xL, BCL2, XIAP, FLICEc-s, dominant negative caspase-8, dominant negative caspase-9, SPI-6 (see, e.g., U.S. Patent Application No. 20070026076), transcription factor PGC-α1, Pinch gene, ILK gene and thymosin 4 gene), coagulation factors (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, intracellular and / or extracellular superoxide dismutase, leptin, LDL receptor, neprilysin, lipoprotein lipase,Ornithine transcarbamylase, β-globulin, α-globulin, spectrin, α1-antitrypsin, methylcytosine binding protein 2, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, β-glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, RP65 protein, cytokines (e.g., interferon α, interferon β, interferon-γ, interleukin-1 to -14, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophic factors and hormones (e.g., growth hormone (somatotropin) in), insulin, insulin-like growth factors including IGF-1 and IGF-2, GLP-1, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophin-3 and -4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factor-α and -β, etc.), bone morphogenetic proteins (including RANKL and VEGF), lysosomal proteins, glutamate receptors, lymphokines, soluble CD4, Fc receptors, T-cell receptors, ApoE, ApoC, protein phosphatase inhibitor 1 inhibitor 1 (I-1), phospholamban, serca2a, lysosomal acid α-glucosidase, α-galactosidase A, Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), calsarcin, receptors (e.g., tumor necrosis growth factor-α soluble receptor), anti-inflammatory factors such as IRAP, Pim-1, PGC-1α, SOD-1, SOD-2, ECF-SOD, kallikrein, thymosin-β4 , hypoxia-inducible transcription factor [HIF], angiogenic factor, S100A1, parvalbumin, type 6 adenylyl cyclase, molecules that affect G protein-coupled receptor kinase type 2 knockout (such as truncated constitutively active bARKct); phospholamban inhibitors or dominant negative molecules such as phospholamban S16E, monoclonal antibodies (including single-chain monoclonal antibodies) or suicide gene products (such as thymidine kinase, cytosine deaminase, diphtheria toxin and tumor necrosis factor such as TNF-α), and any other polypeptides that have a therapeutic effect in a subject in need thereof.

[0148] The heterologous nucleotide sequence of the coded polypeptide includes those encoding reporter polypeptide (e.g., enzyme).Reporter polypeptide is known in the art, including but not limited to fluorescent protein (e.g., EGFP, GFP, RFP, BFP, YFP or dsRED2), enzymes producing detectable products, such as luciferase (e.g., from Gaussia, Renilla or Photinus), β-galactosidase, β-glucuronidase, alkaline phosphatase, and chloramphenicol acetyltransferase gene, or directly detectable protein.Almost any protein can be directly detected by using, for example, a specific antibody of the protein.Sambrook and Russell (2001), Molecular Cloning, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al. (1992), Current Protocols in Molecular Biology, John Wiley & Sons (including regular updates) disclose other markers (and related antibiotics) suitable for positive or negative selection of eukaryotic cells.

[0149] Alternatively, the heterologous nucleic acid can encode a functional RNA, such as an antisense oligonucleotide, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), an RNA that affects spliceosome-mediated trans-splicing (see Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702), an interfering RNA (RNAi), including small interfering RNA (siRNA) that mediates gene silencing (see Sharp et al., (2000) Science 287:2431), a microRNA or other non-translated "functional" RNA, such as a "guide" RNA (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.), and the like. Exemplary non-translated RNAs include RNAi or antisense RNAs to multidrug resistance (MDR) gene products (e.g., for treating tumors and / or administering to the heart to prevent damage from chemotherapy), RNAi or antisense RNAs to myostatin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNAs to VEGF or tumor immunogens (including but not limited to those tumor immunogens specifically described herein) (for treating tumors), RNAi or antisense oligonucleotides to mutant dystrophin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNAs to hepatitis B surface antigen genes (to prevent and / or treat hepatitis B infection), RNAi or antisense RNAs to HIV tat and / or rev genes (to prevent and / or treat HIV), and / or RNAi or antisense RNAs to any other immunogens from pathogens (to protect subjects from pathogens) or defective gene products (to prevent or treat disease). RNAi or antisense RNAs to the above targets or any other targets can also be used as research reagents.

[0150] As is known in the art, antisense nucleic acids (e.g., DNA or RNA) and inhibitory RNA (e.g., microRNA and RNAi such as siRNA or shRNA) sequences can be used to induce "exon skipping" in patients with muscular dystrophy caused by defects in the dystrophin gene. Thus, the heterologous nucleic acid can encode an antisense nucleic acid or inhibitory RNA that induces appropriate exon skipping. It will be appreciated by those skilled in the art that the specific method of exon skipping depends on the nature of the underlying defect in the dystrophin gene, and many such strategies are known in the art. Exemplary antisense nucleic acid and inhibitory RNA sequences target upstream branch points and / or downstream donor splice sites and / or internal splicing enhancer sequences of one or more dystrophin exons (e.g., exon 19 or 23). For example, in a particular embodiment, the heterologous nucleic acid encodes an antisense nucleic acid or inhibitory RNA directed to the upstream branch point and downstream splice donor site of exon 19 or 23 of the dystrophin gene. Such sequences can be incorporated into AAV vectors that deliver modified U7 snRNA and antisense or inhibitory RNA (see, e.g., Goyenvalle et al., (2004) Science 306: 1796-1799). As another strategy, modified U1 snRNA can be incorporated into AAV vectors together with siRNA, microRNA, or antisense RNA complementary to upstream and downstream splice sites of dystrophin exons (e.g., exon 19 or 23) (see, e.g., Denti et al., (2006) Proc. Nat. Acad. Sci. USA 103: 3758-3763). In addition, antisense and inhibitory RNAs can target splicing enhancer sequences within exons 19, 43, 45, or 53 (see, e.g., U.S. Pat. No. 6,653,467; U.S. Pat. No. 6,727,355; and U.S. Pat. No. 6,653,466).

[0151] Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes have a specific catalytic domain that has endonuclease activity (Kim et al., (1987) Proc. Natl. Acad. Sci. USA 84: 8788; Gerlach et al., (1987) Nature 328: 802; Forster and Symons, (1987) Cell 49: 211). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, usually cleaving only one of the multiple phosphoesters in the oligonucleotide substrate (Michel and Westhof, (1990) J. Mol. Biol. 216: 585; Reinhold-Hurek and Shub, (1992) Nature 357: 173). This specificity has been attributed to the need for the substrate to bind to the internal guide sequence ("IGS") of the ribozyme through specific base pairing interactions prior to the chemical reaction.

[0152] Ribozyme catalysis has been observed primarily as part of sequence-specific cleavage / ligation reactions involving nucleic acids (Joyce, (1989) Nature 338:217). For example, U.S. Pat. No. 5,354,855 reports that certain ribozymes can act as endonucleases with sequence specificities greater than those of known ribozymes and approaching those of DNA restriction enzymes. Therefore, sequence-specific ribozyme-mediated inhibition of nucleic acid expression may be particularly useful for therapeutic applications (Scanlon et al., (1991) Proc. Natl. Acad. Sci. USA 88:10591; Sarver et al., (1990) Science 247:1222; Sioud et al., (1992) J. Mol. Biol. 223:831).

[0153] MicroRNA (mir) is a natural cell RNA molecule that can regulate the expression of multiple genes by controlling the stability of mRNA. Overexpression or reduction of specific microRNA can be used to treat dysfunction, and has been shown to be effective in many disease states and animal models of diseases (see, for example, Couzin, (2008) Science 319: 1782-4). Chimeric AAV can be used to deliver microRNA to cells, tissues and subjects for the treatment of genetic and acquired diseases, or for enhancing the functionality and growth promotion of certain tissues. For example, mir-1, mir-133, mir-206 and / or mir-208 can be used to treat heart and skeletal muscle diseases (see, for example, Chen et al., (2006) Genet.38: 228-33; van Rooij et al., (2008) Trends Genet.24: 159-66). MicroRNA can also be used to regulate the immune system after gene delivery (Brown et al., (2007) Blood 110: 4144-52).

[0154] As used herein, the term "antisense oligonucleotide" (including "antisense RNA") refers to a nucleic acid that is complementary to and specifically hybridizes to a specified DNA or RNA sequence. Antisense oligonucleotides and nucleic acids encoding the antisense oligonucleotides can be prepared according to conventional techniques. See, for example, U.S. Pat. No. 5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al.

[0155] Those skilled in the art will understand that the antisense oligonucleotide need not be completely complementary to the target sequence, as long as the degree of sequence similarity is sufficient to enable the antisense nucleotide sequence to specifically hybridize to its target (as defined above) and reduce the production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more).

[0156] To determine the specificity of hybridization, hybridization of such oligonucleotides to target sequences can be performed under reduced stringency, moderate stringency or even stringent conditions. Suitable conditions for achieving reduced, moderate and stringent hybridization conditions are as described herein.

[0157] Alternatively, in specific embodiments, the antisense oligonucleotides of the invention have at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or more sequence identity to the complement of the target sequence, and reduce the production of a protein product (as defined above). In some embodiments, the antisense sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches compared to the target sequence.

[0158] Methods for determining percent nucleic acid sequence identity are described in more detail elsewhere herein.

[0159] The length of the antisense oligonucleotide is not critical, as long as it specifically hybridizes to a predetermined target and reduces the production of a protein product (as defined above) and can be determined according to routine procedures. Typically, the length of the antisense oligonucleotide is at least about 8, 10 or 12 or 15 nucleotides and / or less than about 20, 30, 40, 50, 60, 70, 80, 100 or 150 nucleotides.

[0160] RNA interference (RNAi) is another useful method (e.g., shRNA or siRNA) to reduce the production of protein products. RNAi is a mechanism of post-transcriptional gene silencing, in which double-stranded RNA (dsRNA) corresponding to a target sequence of interest is introduced into a cell or organism, resulting in degradation of the corresponding mRNA. Sharp et al., (2001) Genes Dev 15: 485-490; and Hammond et al., (2001) Nature Rev. Gen. 2: 110-119) reviewed the mechanism of RNAi gene silencing. Before regaining gene expression, the RNAi effect lasts for multiple cell divisions. Therefore, RNAi is a powerful method for targeted knockout or "knockdown" at the RNA level. It has been shown that RNAi is successful in human cells including human embryonic kidney and HeLa cells (see, e.g., Elbashir et al., Nature (2001) 411: 494-8).

[0161] Initial attempts to use RNAi in mammalian cells generated an antiviral defense mechanism involving PKR in response to dsRNA molecules (see, e.g., Gil et al., (2000) Apoptosis 5:107). Short synthetic dsRNAs of about 21 nucleotides, called "short interfering RNAs" (siRNAs), have been shown to mediate silencing in mammalian cells without eliciting an antiviral response (see, e.g., Elbashir et al., Nature (2001) 411:494-8; Caplen et al., (2001) Proc. Nat. Acad. Sci. USA 98:9742).

[0162] RNAi molecules (including siRNA molecules) can be short hairpin RNA (shRNA; see Paddison et al., (2002), Proc. Nat. Acad. Sci. USA 99: 1443-1448), which is considered to be processed into 20-25 chain siRNA molecules in cells by the action of RNase III-like enzyme Dicer. shRNA generally has a stem-loop structure in which two inverted repeat sequences are separated by a short spacer sequence that loops out. shRNAs with loops of 3 to 23 nucleotides in length have been reported. The loop sequence is generally not important. Exemplary loop sequences include the following motifs: AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA.

[0163] RNAi can further comprise a circular molecule containing sense and antisense regions, with two loop regions on one of its sides, so as to form a "dumbbell" shaped structure when dsRNA is formed between the sense and antisense regions. The molecule can be treated in vitro or in vivo to release the dsRNA portion, such as siRNA.

[0164] International Patent Publication WO 01 / 77350 describes a vector for bidirectional transcription to produce sense and antisense transcripts of a heterologous sequence in eukaryotic cells. This technology can be used to generate RNAi for use in accordance with the present invention.

[0165] Shinagawa et al., (2003) Genes Dev. 17: 1340 reported a method for expressing long dsRNA from a CMV promoter (pol II promoter), which is also applicable to tissue-specific pol II promoters. Similarly, the method of Xia et al., (2002) Nature Biotech. 20: 1006 avoids poly (A) tailing and can be used in combination with tissue-specific promoters.

[0166] Methods for producing RNAi include chemical synthesis, in vitro transcription, digestion of long dsRNA by Dicer (in vitro or in vivo), in vivo expression from delivery vectors, and in vivo expression from PCR-derived RNAi expression cassettes (see, e.g., Tech Notes 10(3) "Five Ways to Produce siRNAs," from Ambion, Inc., Austin TX; available at www.ambion.com).

[0167] Guidelines for designing siRNA molecules are available (see, e.g., literature from Ambion, Inc., Austin TX; available at www.ambion.com). In a specific embodiment, the siRNA sequence has a G / C content of about 30-50%. In addition, if RNA polymerase III is used to transcribe the RNA, long stretches of more than four T or A residues are generally avoided. Online siRNA target finders are available, e.g., from Ambion, Inc. (www.ambion.com), through the Whitehead Institute for Biomedical Research (www.jura.wi.mit.edu), or from Dharmacon Research, Inc. (www.dharmacon.com).

[0168] The antisense region of the RNAi molecule can be completely complementary to the target sequence, but as long as it specifically hybridizes to the target sequence (as defined above) and reduces the production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more), it does not need to be completely complementary. In some embodiments, hybridization of such an oligonucleotide to the target sequence can be carried out under reduced stringency, moderate stringency, or even stringent conditions as defined above.

[0169] In other embodiments, the antisense region of the RNAi has at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or more sequence identity to the complementary sequence of the target sequence and reduces the production of the protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, the antisense region contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches compared to the target sequence. Mismatches are generally better tolerated at the ends of the dsRNA than in the central portion.

[0170] In a specific embodiment, RNAi is formed by intermolecular complexation between two separate sense and antisense molecules. RNAi comprises a ds region formed by intermolecular base pairs between two separate strands. In other embodiments, RNAi comprises a ds region formed by intramolecular base pairing within a single nucleic acid molecule comprising sense and antisense regions, typically an inverted repeat (e.g., shRNA or other stem-loop structures, or circular RNAi molecules). RNAi may also comprise a spacer between the sense and antisense regions.

[0171] RNAi molecules are usually highly selective. If desired, those skilled in the art can easily exclude candidate RNAi that may interfere with the expression of nucleic acids other than the target by, for example, using BLAST (available at www.ncbi.nlm.nih.gov / BLAST) to search relevant databases to identify RNAi sequences that do not have substantial sequence homology to other known sequences.

[0172] Kits for producing RNAi are commercially available from, for example, New England Biolabs, Inc. and Ambion, Inc.

[0173] Recombinant viral vectors can also contain heterologous nucleotide sequences that share homology with and recombine with sites on the host chromosome. This approach can be used to correct genetic defects in host cells.

[0174] The present invention also provides recombinant viral vectors expressing immunogenic polypeptides, which are used, for example, for vaccination. The heterologous nucleic acid can encode any immunogen of interest known in the art, including but not limited to immunogens from human immunodeficiency virus, influenza virus, gag protein, tumor antigens, cancer antigens, bacterial antigens, viral antigens, etc. Alternatively, the immunogen can be present in (e.g., incorporated into) or connected to (e.g., by covalent modification) the viral capsid.

[0175] The use of parvovirus as a vaccine is known in the art (see, e.g., Miyamura et al., (1994) Proc. Nat. Acad. Sci. USA 91:8507; U.S. Pat. No. 5,916,563 to Young et al., U.S. Pat. No. 5,905,040 to Mazzara et al., U.S. Pat. No. 5,882,652, U.S. Pat. No. 5,863,541 to Samulski et al.; the disclosures of which are incorporated herein by reference in their entirety). The antigen may be present in the viral capsid. Alternatively, the antigen may be expressed by a heterologous nucleic acid introduced into the genome of a recombinant vector.

[0176] The immunogenic polypeptide or immunogen can be any polypeptide suitable for protecting a subject from a disease, including but not limited to microbial, bacterial, protozoan, parasitic, fungal and viral diseases. For example, the immunogen can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus hemagglutinin (HA) surface protein or an influenza virus nucleoprotein gene, or an equine influenza virus immunogen), or a lentivirus immunogen (e.g., an equine infectious anemia virus immunogen, a simian immunodeficiency virus (SIV) immunogen, or a human immunodeficiency virus (HIV) immunogen, such as HIV or SIV envelope GP160 protein, HIV or SIV matrix / capsid protein, and HIV or SIV gag, pol and env gene products). The immunogen can also be an arenavirus immunogen (e.g., a Lassa fever virus immunogen, such as a Lassa fever virus nucleocapsid protein gene and a Lassa fever envelope glycoprotein gene), a poxvirus immunogen (e.g., vaccinia, such as vaccinia L1 or L8 gene), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus immunogen or a Marburg virus immunogen, such as NP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF and SFS viruses), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as a human coronavirus envelope glycoprotein gene, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen, or a severe acute respiratory syndrome (SARS) immunogen such as S [S1 or S2], M, E or N protein or an immunogenic fragment thereof). The immunogen may further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogens), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, or hepatitis C) immunogen, or any other vaccine immunogen known in the art.

[0177] Alternatively, the immunogen can be any tumor or cancer cell antigen. Alternatively, the tumor or cancer antigen is expressed on the surface of a cancer cell. Exemplary cancer and tumor cell antigens are described in A. Rosenberg, (1999) Immunity 10: 281).Illustrative cancer and tumor antigens include, but are not limited to, BRCA1 gene products, BRCA2 gene products, gp100, tyrosinase, GAGE-1 / 2, BAGE, RAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et al., (1994) Cancer Res. 54:3124) including MART-1 (Coulie et al., (1991) J. Exp. Med. 180:35), gp100 (Wick et al., (1994) J. Exp. Med. 180:35), al., (1988) J. Cutan. Pathol. 4:201) and MAGE antigens (MAGE-1, MAGE-2 and MAGE-3) (Van der Bruggen et al., (1991) Science, 254:1643), CEA, TRP-1; TRP-2; P-15 and tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER-2 / neu gene product (U.S. Pat. No. 4,968,603); CA125; HE4; LK26; FB5 (endosialin); TAG 72; AFP; CA19-9; NSE; DU-PAN-2; CA50; Span-1; CA72-4; HCG; STN (sialyl Tn antigen); c-erbB-2 protein; PSA; L-CanAg; estrogen receptor; milk fat globulin; p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62: 623); mucin antigen (International Patent Publication No. WO 90 / 05142); telomerase; nuclear matrix protein; prostatic acid phosphatase; papillomavirus antigens; and antigens associated with the following cancers: melanoma, adenocarcinoma, thymoma, sarcoma, lung cancer, liver cancer, colorectal cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, etc. (see, e.g., Rosenberg, (1996) Annu. Rev. Med. 47:481-91).

[0178] Alternatively, the heterologous nucleotide sequence may encode any polypeptide that is desired to be produced in cells in vitro, ex vivo or in vivo. For example, a viral vector may be introduced into cultured cells and the expressed protein product isolated therefrom.

[0179] Those skilled in the art will appreciate that the heterologous polynucleotide of interest can be operably associated with appropriate control sequences. For example, the heterologous nucleic acid can be operably associated with expression control elements (such as transcription / translation control signals, replication origins, polyadenylation signals, internal ribosome entry sites (IRES), promoters, enhancers, etc.).

[0180] Those skilled in the art will further appreciate that various promoter / enhancer elements may be used, depending on the desired level and tissue-specific expression. Depending on the desired expression pattern, the promoter / enhancer may be constitutive or inducible. The promoter / enhancer may be native or foreign, and may be a native or synthetic sequence. "Foreign" refers to a transcriptional initiation region that is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0181] The promoter / enhancer element can be native to the target cell or subject to be treated, and / or native to the heterologous nucleic acid sequence. The promoter / enhancer element is generally selected so that it functions in the target cell of interest. In representative embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhancer element can be constitutive or inducible.

[0182] Inducible expression control elements are generally used in those applications where it is necessary to provide regulation of heterologous nucleic acid sequence expression. The inducible promoter / enhancer element for gene delivery can be tissue-specific or tissue-preferred promoter / enhancer elements, and include muscle specificity or preference (including heart, skeleton and / or smooth muscle), neural tissue specificity or preference (including brain specificity), eyes (including retina specificity and cornea specificity), liver specificity or preference, bone marrow specificity or preference, pancreas specificity or preference, spleen specificity or preference and lung specificity or preference promoter / enhancer elements. Other inducible promoter / enhancer elements include hormone inducible and metal inducible elements. Exemplary inducible promoter / enhancer elements include but are not limited to Tet on / off element, RU486 inducible promoter, ecdysone inducible promoter, rapamycin inducible promoter and metallothionein promoter.

[0183] In embodiments where a heterologous nucleic acid sequence is transcribed and translated in a target cell, specific initiation signals are generally used for efficient translation of the inserted protein coding sequence. These exogenous translation control sequences, which may include the ATG initiation codon and adjacent sequences, may be of a variety of origins (natural and synthetic).

[0184] The present invention also provides a method for producing a viral vector of the present invention. In a representative embodiment, the present invention provides a method for producing a recombinant viral vector, the method comprising providing (a) a template to a cell in vitro, the template comprising (i) a polynucleotide of interest and (ii) a packaging signal sequence (e.g., one or more (e.g., two) terminal repeats, such as AAV terminal repeats) sufficient to encapsulate the AAV template into a viral particle, and (b) an AAV sequence (e.g., AAV rep and AAV cap sequences) sufficient to replicate the template and load the template into the viral particle. Under conditions such that a recombinant viral particle comprising a template packaged in a capsid is produced in the cell, the template and the AAV replication and capsid sequences are provided. The method may also include a step of collecting viral particles from the cell. Viral particles may be collected from the culture medium and / or by lysing cells.

[0185] In an illustrative embodiment, the invention provides a method for producing rAAV particles comprising an AAV capsid, the method comprising: providing to cells in vitro a nucleic acid encoding an AAV capsid, an AAV rep coding sequence, an AAV vector genome comprising a polynucleotide of interest, and helper functions for producing a productive AAV infection; and allowing assembly of AAV particles comprising the AAV capsid and encapsidating the AAV vector genome.

[0186] The cells are typically cells that are permissive for AAV viral replication. Any suitable cell known in the art may be used, such as a mammalian cell. Also suitable are trans-complementing packaging cell lines that provide functions missing from the replication-defective helper virus, such as 293 cells or other E1a trans-complementing cells.

[0187] AAV replication and capsid sequences can be provided by any method known in the art. Current protocols typically express AAV rep / cap genes on a single plasmid. It is not necessary to provide AAV replication and packaging sequences together, although it may be convenient to do so. AAV rep and / or cap sequences can be provided by any viral vector or non-viral vector. For example, the rep / cap sequence can be provided by a hybrid adenovirus or herpes virus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus vector). EBV vectors can also be used to express AAV cap and rep genes. One advantage of this method is that EBV vectors are episomal, but will maintain a high copy number throughout continuous cell divisions (i.e., stably integrated into the cell as an extrachromosomal element, designated as an EBV-based nuclear episome).

[0188] As another alternative, the rep / cap sequences may be stably harbored within the cell (either episomal or integrated).

[0189] Typically, the AAV rep / cap sequences are not flanked by AAV packaging sequences (eg, AAV ITRs) to prevent rescue and / or packaging of these sequences.

[0190] The template (e.g., rAAV vector genome) can be provided to the cell using any method known in the art. For example, the template can be provided by a non-viral (e.g., plasmid) or viral vector. In a specific embodiment, the template is provided by a herpes virus or an adenovirus vector (e.g., inserted into the E1a or E3 region of a missing adenovirus). As another illustration, Palombo et al., (1998) J. Virol. 72: 5025 describes a baculovirus vector carrying a reporter gene flanked by AAV ITR. The template can also be delivered using an EBV vector, as described above for the rep / cap gene.

[0191] In another representative embodiment, the template is provided by a replicating rAAV virus. In yet other embodiments, the AAV provirus is stably integrated into the chromosome of the cell.

[0192] In order to obtain maximum viral titer, cells are usually provided with helper virus functions (e.g., adenovirus or herpes virus) that are important for productive AAV infection. Helper virus sequences required for AAV replication are known in the art. Typically, these sequences are provided by helper adenovirus or herpes virus vectors. Alternatively, adenovirus or herpes virus sequences can be provided by another non-viral or viral vector, for example, as a non-infectious adenovirus miniplasmid carrying all the helper genes required for effective AAV production, as described in Ferrari et al., (1997) Nature Med. 3: 1295 and U.S. Patent Nos. 6,040,183 and 6,093,570.

[0193] In addition, helper virus function can be provided by a packaging cell that has a helper gene integrated into the chromosome or maintained as a stable extrachromosomal element. In a representative embodiment, the helper virus sequence cannot be packaged in an AAV virion, e.g., is not flanked by AAV ITRs.

[0194] Those skilled in the art will appreciate that it may be advantageous to provide AAV replication and capsid sequences and helper virus sequences (e.g., adenovirus sequences) on a single helper construct. The helper construct may be a non-viral or viral construct, but may alternatively be a hybrid adenovirus or hybrid herpes virus containing the AAV rep / cap genes.

[0195] In a specific embodiment, the AAV rep / cap sequence and the adenovirus helper sequence are provided by a single adenovirus helper vector. The vector also contains a rAAV template. The AAV rep / cap sequence and / or the rAAV template can be inserted into a deleted region of the adenovirus (e.g., E1a or E3 region).

[0196] In another embodiment, the AAV rep / cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector. The rAAV template is provided as a plasmid template.

[0197] In another illustrative embodiment, the AAV rep / cap sequences and adenovirus helper sequences are provided by a single adenovirus helper vector, and the rAAV template is integrated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector maintained in the cell as an extrachromosomal element (e.g., as an "EBV-based nuclear episome," see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).

[0198] In another exemplary embodiment, the AAV rep / cap sequence and the adenovirus helper sequence are provided by a single adenovirus helper. The rAAV template is provided as a separate replicating viral vector. For example, the rAAV template can be provided by a rAAV particle or a second recombinant adenovirus particle.

[0199] According to the above method, the hybrid adenoviral vector generally comprises adenoviral 5' and 3' cis sequences (i.e., adenoviral terminal repeats and PAC sequences) sufficient for adenoviral replication and packaging. The AAV rep / cap sequence and (if present) the rAAV template are embedded in the adenoviral backbone and are flanked by the 5' and 3' cis sequences so that these sequences can be packaged into the adenoviral capsid. As described above, in representative embodiments, the adenoviral helper sequence and the AAV rep / cap sequence are not flanked by the AAV packaging sequence (e.g., AAV ITR) so that these sequences are not packaged into the AAV virion.

[0200] Herpes viruses can also be used as helper viruses in AAV packaging methods. Hybrid herpes viruses encoding AAV rep proteins can advantageously promote more scalable AAV vector production schemes. Hybrid herpes simplex virus type I (HSV-1) vectors expressing AAV-2 rep and cap genes have been described (Conway et al., (1999) Gene Therapy 6:986 and WO 00 / 17377, the disclosures of which are incorporated herein in their entirety).

[0201] As another alternative, the viral vectors of the invention can be produced in insect cells using baculovirus vectors to deliver the rep / cap genes and the rAAV template as described by Urabe et al., (2002) Human Gene Therapy 13: 1935-43.

[0202] Other methods of producing AAV use stably transformed packaging cells (see, e.g., U.S. Pat. No. 5,658,785).

[0203] AAV vector stocks without contaminating helper viruses can be obtained by any method known in the art. For example, AAV and helper viruses can be easily distinguished according to size. AAV can also be separated from helper viruses based on affinity for heparin substrates (Zolotukhin et al., (1999) Gene Therapy 6:973). In a representative embodiment, a deleted replication-deficient helper virus is used so that any contaminating helper virus does not have replication ability. As another alternative, an adenovirus helper lacking late gene expression can be used, because only adenovirus early gene expression is required to mediate the packaging of AAV viruses. Adenovirus mutants defective in late gene expression are known in the art (e.g., ts100K and ts149 adenovirus mutants).

[0204] The packaging method of the present invention can be used to produce a high titer virus particle stock. In a specific embodiment, the virus stock has a titer of at least about 10 5 transduction units (tu) / ml, at least about 10 6 tu / ml, at least about 10 7 tu / ml, at least about 10 8 tu / ml, at least about 10 9 tu / ml or at least about 10 10 tu / ml.

[0205] In a specific embodiment, the present invention provides a pharmaceutical composition comprising a viral vector of the present invention and optional other medical agents, medicaments, stabilizers, buffers, carriers, adjuvants, diluents, etc. in a pharmaceutically acceptable carrier. For injection, the carrier is generally a liquid. For other modes of administration, the carrier can be solid or liquid. For inhalation administration, the carrier will be respirable and will preferably be in the form of solid or liquid particles.

[0206] The term "pharmaceutically acceptable" refers to a substance that is not toxic or otherwise undesirable, ie, the substance can be administered to a subject without causing any undesirable biological effects.

[0207] One aspect of the invention is a method for transferring a polynucleotide of interest to a cell in vitro. The viral vector can be introduced into the cell at an appropriate multiplicity of infection according to standard transduction methods suitable for the particular target cell. The titer of the viral vector or capsid for administration can vary depending on the type and number of target cells and the particular viral vector or capsid, and can be determined by one skilled in the art without undue experimentation. In a specific embodiment, at least about 10 3 infectious units, more preferably at least about 10 5 infectious unit.

[0208] The cell that can introduce viral vector can be any type, including but not limited to neural cell (including peripheral and central nervous system cells, particularly, brain cells such as neurons, oligodendrocytes, glial cells, astrocytes), lung cells, eye cells (including retinal cells, retinal pigment epithelium and corneal cells), epithelial cells (such as intestinal and respiratory epithelial cells), skeletal muscle cells (including myoblasts, myotubes and myofibers), diaphragm muscle cells, dendritic cells, pancreatic cells (including islet cells), hepatocytes, gastrointestinal cells (including smooth muscle cells, epithelial cells), heart cells (including cardiomyocytes), bone cells (such as bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, joint cells (including such as cartilage, meniscus, synovium and bone marrow), germ cells etc. Alternatively, the cell can be any progenitor cell. As an alternative, the cell can be a stem cell (such as neural stem cell, liver stem cell). As another alternative, the cell can be a cancer or tumor cell (cancer and tumor as described above). Furthermore, as noted above, the cells may be from any species of origin.

[0209] Viral vectors can be introduced into cells in vitro, with the purpose of administering modified cells to a subject. In a specific embodiment, cells have been removed from a subject, viral vectors are introduced therein, and then the cells are replaced back into the subject. Cells are taken out from a subject for in vitro treatment and then introduced back into the subject's method as known in the art (see, e.g., U.S. Patent No. 5,399,346). Alternatively, recombinant viral vectors are introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need.

[0210] Suitable cells for ex vivo gene therapy are described above. The dosage of cells administered to a subject will vary depending on the age, condition and species of the subject, the cell type, the nucleic acid expressed by the cell, the mode of administration, etc. Typically, at least about 10 μg of the cell per dose will be administered in a pharmaceutically acceptable carrier. 2 to about 10 8 or about 10 3 to about 10 6 In a specific embodiment, cells transduced with a viral vector are administered to a subject in an effective amount in combination with a pharmaceutical carrier.

[0211] In some embodiments, cells that have been transduced with a viral vector can be administered to elicit an immunogenic response to the delivered polypeptide (e.g., expressed as a transgene or in a capsid). Typically, a certain amount of cells expressing an effective amount of a polypeptide in combination with a pharmaceutically acceptable carrier is administered. Alternatively, the dose is sufficient to produce a protective immune response (as defined above). As long as the benefits of administering an immunogenic polypeptide outweigh any disadvantages thereof, the degree of protection conferred does not need to be complete or permanent.

[0212] Another aspect of the present invention is a method of administering a viral vector of the present invention to a subject. In a specific embodiment, the method comprises a method of delivering a polynucleotide of interest to an animal subject, the method comprising: administering an effective amount of a viral vector according to the present invention to an animal subject. The viral vector of the present invention can be administered to a human subject or animal in need thereof by any method known in the art. Alternatively, a viral vector in a pharmaceutically acceptable carrier is delivered in an effective dose.

[0213] The viral vector of the present invention can be further administered to a subject to induce an immunogenic response (e.g., as a vaccine). Typically, the vaccine of the present invention comprises an effective amount of virus in combination with a pharmaceutically acceptable carrier. Alternatively, the dosage is sufficient to produce a protective immune response (as defined above). As long as the benefits of administering the immunogenic polypeptide outweigh any of its disadvantages, the degree of protection conferred does not need to be complete or permanent. The subject and the immunogen are as described above.

[0214] The dosage of the viral vector to be administered to a subject will depend on the mode of administration, the disease or condition to be treated, the condition of the individual subject, the particular viral vector and the nucleic acid to be delivered, and can be determined in a routine manner. An exemplary dosage for achieving a therapeutic effect is at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 3 , 10 14 , 10 15 The virus titer should be about 10 transducing units or higher, preferably about 10 7 or 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 transduction units, and more preferably about 10 12transduction units.

[0215] In specific embodiments, more than one administration (eg, two, three, four, or more administrations) may be used to achieve desired levels of gene expression over various intervals of time (eg, daily, weekly, monthly, yearly, etc.).

[0216] Exemplary modes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., by aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to bones, diaphragm and / or myocardium], intradermal, intrapleural, intracerebral and intraarticular), topical (e.g., skin and mucosal surfaces, including airway surfaces and transdermal administration), intralymphatic, etc., and direct tissue or organ injection (e.g., liver, skeletal muscle, myocardium, diaphragm muscle or brain). Administration to tumors (e.g., in or near a tumor or lymph node) is also possible. The most appropriate route in any given case will depend on the nature and severity of the condition being treated and the nature of the particular carrier being used.

[0217] Delivery to any of these tissues can also be achieved by delivering a depot comprising a viral vector, which can be implanted in the tissue or the tissue can be contacted with a membrane or other matrix comprising the viral vector. Examples of such implantable matrices or substrates are described in U.S. Patent No. 7,201,898.

[0218] The present invention can be used for treating the disease of tissue or organ.Alternately, the present invention can be implemented to deliver nucleic acid to tissue or organ, described tissue or organ is used as for producing and usually circulates in blood or systemically delivers to other tissues to treat disease (for example metabolic disorder, such as diabetes (for example insulin), hemophilia (for example factor IX or factor VIII) or lysosomal storage disorder (such as Gaucher's disease [glucocerebrosidase], Pompe disease [lysosomal acid α-glucosidase] or Fabry disease [α-galactosidase A]) or glycogen storage disease (such as Pompe disease [lysosomal acid α-glucosidase]) protein product (for example enzyme) or non-translated RNA (for example RNAi, microRNA, antisense RNA) platform.Other suitable proteins for treating metabolic disorder are as described above.

[0219] Injectables can be prepared in conventional forms, as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquids before injection, or as emulsions. Alternatively, viral vectors can be administered in a local rather than systemic manner, for example in the form of a reservoir or a sustained-release formulation. In addition, viral vectors can be delivered dry to surgical implantable matrices, such as bone graft substitutes, sutures, stents, etc. (e.g., as described in U.S. Patent No. 7,201,898).

[0220] Pharmaceutical compositions suitable for oral administration may be present in discrete units, such as capsules, cachets, lozenges or tablets, each of which contains a predetermined amount of the composition of the present invention; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as a water-in-oil or water-in-oil emulsion. Oral delivery can be performed by compounding the viral vector of the present invention with a carrier that can withstand degradation by digestive enzymes in the animal intestine. Examples of such carriers include plastic capsules or tablets known in the art. These preparations are prepared by any suitable pharmaceutical method, including the step of combining the composition with a suitable carrier (the carrier may contain one or more auxiliary ingredients as described above). Typically, a pharmaceutical composition according to an embodiment of the present invention is prepared by uniformly and intimately mixing the composition with a liquid or finely divided solid carrier or both, and then molding the resulting mixture if necessary. For example, tablets can be prepared by compressing or molding a powder or granules containing the composition and optionally one or more auxiliary ingredients. Compressed tablets are prepared by compressing a free-flowing composition (such as a powder or granules optionally mixed with a binder, a lubricant, an inert diluent and / or a surfactant / dispersant) in a suitable machine. Molded tablets are made by molding in a suitable machine the powdered compound moistened with an inert liquid binder.

[0221] Pharmaceutical compositions suitable for buccal (sublingual) administration include lozenges comprising the compositions of this invention in a flavored base, usually sucrose and acacia or tragacanth, and pastilles comprising the compositions in an inert base such as gelatin and glycerin or sucrose and acacia.

[0222] Pharmaceutical compositions suitable for parenteral administration may include sterile aqueous and non-aqueous injections of the compositions of the present invention, which are optionally isotonic with the blood of the intended recipient. These preparations may contain antioxidants, buffers, antibacterial agents and solutes, which make the compositions isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions, solutions and emulsions may include suspending agents and thickening agents. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / water solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oil. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (e.g., those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents and inert gases, etc.

[0223] The compositions may be presented in unit / dose or multi-dose containers, for example in sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, for example saline or water for injection, immediately prior to use.

[0224] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the above types. For example, the injectable stable sterile composition of the present invention can be provided in a unit dosage form in a sealed container. The composition can be provided in the form of a lyophilized product, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The unit dosage form can be about 1 μg to about 10g of the composition of the present invention. When the composition is substantially insoluble in water, a sufficient amount of physiologically acceptable emulsifier can be included in an aqueous carrier in sufficient amount to emulsify the composition. One such useful emulsifier is phosphatidylcholine.

[0225] Pharmaceutical compositions suitable for rectal administration may be presented as unit-dose suppositories. These may be prepared by mixing the composition with one or more conventional solid carriers (eg, cocoa butter) and then shaping the resulting mixture.

[0226] The pharmaceutical composition of the present invention suitable for topical application to the skin can be in the form of an ointment, cream, lotion, paste, gel, spray, aerosol or oil. The carrier that can be used includes but is not limited to vaseline, lanolin, polyethylene glycol, alcohol, transdermal enhancer and a combination of two or more thereof. In some embodiments, for example, the pharmaceutical composition of the present invention can be mixed with a lipophilic agent (e.g., DMSO) that can enter the skin for topical delivery.

[0227] The pharmaceutical composition suitable for transdermal administration can be in the form of a dispersible patch, which is suitable for keeping close contact for a long time with the epidermis of the experimenter. Compositions suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharm. Res. 3: 318 (1986)) and generally adopt the form of an aqueous solution of the composition of the present invention of optional buffering. Suitable preparations can include citrate or bis\tris buffer (pH 6) or ethanol / water, and can contain 0.1 to 0.2M active ingredient.

[0228] The viral vectors disclosed herein can be administered to the lungs of a subject in any suitable manner, for example, by administering an aerosol suspension of respirable particles composed of the viral vector, which is inhaled by the subject. The inhalable particles can be liquid or solid. As known to those skilled in the art, an aerosol of liquid particles containing viral vectors can be produced by any suitable device, such as a pressure-driven nebulizer or ultrasonic nebulizer. See, for example, U.S. Patent No. 4,501,729. An aerosol of solid particles containing viral vectors can also be produced by any solid particulate drug aerosol generator using techniques known in the pharmaceutical field.

[0229] Production of Factor VIII Protein of the Invention

[0230] Many expression vectors can be used to create genetically engineered cells. Some expression vectors are designed to express large amounts of recombinant proteins after transfected cells are amplified under various conditions that favor selected high-expressing cells. Some expression vectors are designed to express large amounts of recombinant proteins without the need for amplification under selective pressure. The present invention includes the production of genetically engineered cells according to standard methods in the art and is independent of the use of any specific expression vector or expression system.

[0231] In order to produce genetically engineered cells to produce large amounts of FVIII protein, cells are transfected with expression vectors containing polynucleotides (e.g., cDNA) encoding the protein. In some embodiments, the FVIII protein is expressed with a selected co-transfected enzyme that causes the FVIII protein to undergo appropriate post-translational modifications in a given cell system.

[0232] The cells may be derived from a variety of sources, but in other aspects may be cells transfected with an expression vector containing a nucleic acid molecule (eg, cDNA) encoding a FVIII protein.

[0233] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology within the skill of the art. Such techniques are fully described in the literature. See, for example, Sambrook, et al., Molecular Cloning; A Laboratory Manual , 2nd edition (1989); DNA Cloning , Volumes I and II (edited by D. N. Glover, 1985); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins, ed., 1984); Transcription and Translation (B.D. Hames & S.J. Higgins, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1986); Immobilized Cells and Enzymes (IRL Press,1986);B.Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Academic Press, Inc.), especially Volumes 154 and 155 (edited by Wu and Grossman and Wu, respectively); Gene Transfer Vectors for Mammalian Cells (Edited by JHMiller and MPCalos, 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology , edited by Mayerand Walker (Academic Press, London, 1987); Scopes, Protein Purification: Principles and Practice , 2nd edition, 1987 (Springer-Verlag, NY); and Handbook of Experimental Immunology Volumes I-IV (DM Weir and CC Blackwell, eds., 1986). All patents, patent applications, and publications cited in this specification are incorporated herein by reference in their entirety.

[0234] Genetic Engineering Technology

[0235] The production of cloned genes, recombinant DNA, vectors, transformed cells, proteins and protein fragments by genetic engineering is well known. See, for example, U.S. Patent No. 4,761,371 to Bell et al., column 6, line 3 to column 9, line 65; U.S. Patent No. 4,877,729 to Clark et al., column 4, line 38 to line 76; U.S. Patent No. 4,912,038 to Schilling, column 3, line 26 to column 14, line 12; and U.S. Patent No. 4,879,224 to Wallner, column 6, line 8 to column 8, line 59.

[0236] Vector is a reproducible DNA construct. Vector is used to increase the nucleic acid of encoding FVIII protein and / or express the nucleic acid of encoding FVIII protein in this article. Expression vector is a reproducible nucleic acid construct, wherein the nucleotide sequence of encoding FVIII protein is operably connected to a suitable control sequence, and this control sequence can realize the expression of nucleotide sequence in a suitable host cell to produce FVIII protein. The needs of this control sequence will change according to the selected host cell and the selected transformation method. Usually, control sequence includes transcription promoter, the optional operator sequence for controlling transcription, the sequence of encoding suitable mRNA ribosome binding site and the sequence of controlling transcription and translation termination.

[0237] Vectors include plasmids, viruses (e.g., AAV, adenovirus, cytomegalovirus), phages, and integrable DNA fragments (i.e., fragments that can be integrated into the host cell genome by recombination). Vectors can replicate and function independently of the host cell genome (e.g., by transient expression), or can be integrated into the host cell genome itself (e.g., stably integrated). Expression vectors can contain promoters and RNA binding sites that are operably linked to the nucleic acid molecule to be expressed and can operate in host cells and / or organisms.

[0238] When DNA regions or nucleotide sequences are functionally related to each other, they are operably linked or operably associated. For example, if a promoter controls the transcription of a coding sequence, then a promoter is operably linked to the coding sequence; or if a ribosome binding site is positioned to allow translation of a coding sequence, then the ribosome binding site is operably linked to the coding sequence.

[0239] Suitable host cells include prokaryotes, yeast or higher order eukaryotic cells such as mammalian cells and insect cells. Cells derived from multicellular organisms are particularly suitable hosts for recombinant FVIII protein synthesis, and mammalian cells are particularly preferred. Propagation of such cells in cell culture has become routine procedure ( Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973)). Examples of useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines and WI138, HEK 293, BHK, COS-7, CV and MDCK cell lines. Expression vectors for such cells typically include, if necessary, an origin of replication, a promoter located upstream of and operably associated with the nucleotide sequence encoding the FVIII protein to be expressed, and a ribosome binding site, an RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcription termination sequence. In one embodiment, expression can be performed in Chinese hamster ovary (CHO) cells using the expression system of U.S. Pat. No. 5,888,809, which is incorporated herein by reference in its entirety.

[0240] Transcriptional and translational control sequences in expression vectors used in transformed vertebrate cells are often provided by viral sources. Non-limiting examples include promoters derived from polyoma virus, adenovirus 2, and simian virus 40 (SV40). See, e.g., U.S. Pat. No. 4,599,308.

[0241] The replication origin may be provided by constructing the vector to include an exogenous source, such as may be derived from SV 40 or other viral (e.g., polyoma virus, adenovirus, VSV or BPV) sources, or may be provided by the host cell chromosomal replication machinery. If the vector is integrated into the host cell chromosome, the host cell chromosome is usually sufficient.

[0242] In addition to using vectors containing viral origins of replication, mammalian cells can also be transformed by co-transformation using a selective marker and a nucleic acid molecule encoding the FVIII protein. Non-limiting examples of suitable selective markers are dihydrofolate reductase (DHFR) or thymidine kinase. This method is further described in U.S. Patent No. 4,399,216, which is incorporated herein by reference in its entirety.

[0243] Other methods suitable for adaptation to synthesize FVIII protein in recombinant vertebrate cell culture include those described in Gething et al. Nature 293:620 (1981); Mantei et al. Nature 281:40; and Levinson et al., EPO Application Nos. 117,060A and 117,058A, each of which is incorporated herein by reference in its entirety.

[0244] Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as baculovirus expression vectors (e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) can be used to practice the present invention, as described in U.S. Pat. Nos. 4,745,051 and 4,879,236 to Smith et al. In general, baculovirus expression vectors comprise a baculovirus genome containing a nucleotide sequence to be expressed, the nucleotide sequence being inserted into the polyhedrin gene at a position within the range of the polyhedrin transcription start signal to the ATG start site and under the transcriptional control of the baculovirus polyhedrin promoter.

[0245] Prokaryotic host cells include gram-negative or gram-positive organisms, such as Escherichia coli (E.coli) or rod-shaped bacteria, respectively. Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Exemplary bacterial host cells are Escherichia coli W3110 (ATCC 27,325), Escherichia coli B, Escherichia coli X1776 (ATCC 31,537) and Escherichia coli 294 (ATCC 31,446). A variety of suitable prokaryotic and microbial vectors can be used. Escherichia coli is usually transformed using pBR322. The most commonly used promoters in recombinant microbial expression vectors include β-lactamase (penicillinase) and lactose promoter systems (Chang et al. Nature 275: 615 (1978); and Goeddel et al. Nature 281: 544 (1979)), tryptophan (trp) promoter system (Goeddel et al. Nucleic Acids Res. 8: 4057 (1980) and EPO Application Publication No. 36,776), and tac promoter (De Boer et al. Proc. Natl. Acad. Sci. USA 80: 21 (1983)). The promoter and Shine-Dalgarno sequence (for prokaryotic host expression) are operably linked to the nucleic acid encoding the FVIII protein, i.e., they are positioned to promote transcription of the FVIII messenger RNA from the DNA.

[0246] Eukaryotic microorganisms such as yeast cultures can also be transformed with protein encoding vectors (see, e.g., U.S. Patent No. 4,745,057). Saccharomyces cerevisiae is the most commonly used of lower eukaryotic host microorganisms, although many other strains are also commonly available. Yeast vectors can contain a replication origin or autonomously replicating sequence (ARS) from a 2 micron yeast plasmid, a promoter, a nucleic acid encoding the FVIII protein, a sequence for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7 (Stinchcomb et al. Nature 282: 39 (1979); Kingsman et al. Gene 7: 141 (1979); Tschemper et al. Gene 10: 157 (1980)). Suitable promoter sequences for yeast vectors include promoters for metallothioneins, 3-phosphoglycerate kinase (Hitzeman et al. J. Biol. Chem. 255:2073 (1980), or other glycolytic enzymes (Hess et al. J. Adv. Enzyme Reg. 7:149 (1968); and Holland et al. Biochemistry 17:4900 (1978)). Vectors and promoters suitable for yeast expression are further described in R. Hitzeman et al., EPO Publication No. 73,657.

[0247] The cloned coding sequences of the present invention can encode FVIII of any species, including mice, rats, dogs, opossums, rabbits, cats, pigs, horses, sheep, cows, guinea pigs, opossums, platypuses and humans, but preferably encode human FVIII proteins. Nucleic acids encoding FVIII that can hybridize with nucleic acids encoding proteins disclosed herein are also included. Hybridization of such sequences with nucleic acids encoding FVIII proteins disclosed herein can be performed in standard in situ hybridization assays under reduced stringency conditions or even under stringent conditions (e.g., stringent conditions represented by washing stringency of 0.3 M NaCl, 0.03 M sodium citrate, 0.1% SDS at 60° C. or even 70° C.). See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989) Cold Spring Harbor Laboratory).

[0248] The FVIII protein produced according to the present invention can be expressed in transgenic animals by known methods. See, for example, U.S. Patent No. 6,344,596, the entire contents of which are incorporated herein by reference. In short, transgenic animals can include, but are not limited to, farm animals (e.g., pigs, goats, sheep, cows, horses, rabbits, etc.), rodents (e.g., mice, rats, and guinea pigs), and domestic pets (e.g., cats and dogs). In some embodiments, livestock such as pigs, sheep, goats, and cows are particularly preferred.

[0249] The transgenic animals of the present invention are produced by introducing into a single-cell embryo a suitable polynucleotide encoding the human FVIII protein of the present invention in such a way that the polynucleotide is stably integrated into the DNA of the germline cells of the mature animal and inherited in a normal Mendelian manner. The transgenic animals of the present invention will have a phenotype of producing FVIII protein in body fluids and / or tissues. The FVIII protein can be removed from these fluids and / or tissues and processed, for example, for therapeutic purposes. (See, for example, Clark et al. "Expression of human anti-hemophilic factor IX in the milk of transgenic sheep" Bio / Technology 7: 487-492 (1989); Van Cott et al. "Haemophilic factors produced by transgenic livestock: abundance can enable alternative therapies worldwide" Haemophilia 10 (4): 70-77 (2004), the entire contents of which are incorporated herein by reference).

[0250] DNA molecules can be introduced into embryos by a variety of means, including but not limited to microinjection, calcium phosphate-mediated precipitation, liposome fusion, or retroviral infection of totipotent or pluripotent stem cells. The transformed cells are then introduced into the embryo and incorporated therein to form a transgenic animal. For example, L. M. Houdebine's Transgenic Animal Generation and UseMethods for making transgenic animals are described in , Harwood Academic Press, 1997. Methods using nuclear transfer or cloning of embryonic or adult cell lines can also produce transgenic animals, as described, for example, in Campbell et al., Nature 380:64-66 (1996) and Wilmut et al., Nature 385:810-813 (1997). In addition, techniques using cytoplasmic injection of DNA can be used, as described in U.S. Pat. No. 5,523,222.

[0251] Transgenic animals producing FVIII can be obtained by introducing a chimeric construct comprising a FVIII coding sequence. Methods for obtaining transgenic animals are well known. See, for example, Hogan et al., MANIPULATING THE MOUSE EMBRYO , (Cold Spring Harbor Press 1986); Krimpenfort et al., Bio / Technology 9:88 (1991); Palmiter et al., Cell 41:343 (1985), Kraemer et al., GENETIC MANIPULATION OF THE EARLY MAMMALIAN EMBRYO , (Cold Spring Harbor Laboratory Press 1985); Hammer et al., Nature 315:680 (1985); Wagner et al., U.S. Patent No. 5,175,385; Krimpenfort et al., U.S. Patent No. 5,175,384, Janne et al., Ann. Med. 24:273 (1992), Bremet al., Chim. Oggi. 11:21 (1993), Clark et al., U.S. Patent No. 5,476,995, the entire contents of which are incorporated herein by reference.

[0252] In some embodiments, cis-acting regulatory regions that are "active" in mammary tissue may be used because the promoter is more active in mammary tissue than in other tissues under the physiological conditions of synthetic milk. These promoters include, but are not limited to, short and long whey acid protein (WAP), short and long α, β and κ caseins, α-lactalbumin and β-lactoglobulin ("BLG") promoters. Signal sequences that secrete the expressed protein directly into other body fluids, particularly blood and urine, may also be used in accordance with the present invention. Examples of these sequences include signal peptides for secreted coagulation factors, including signal peptides for FVIII, protein C, and tissue plasminogen activator.

[0253] In addition to the promoters discussed above, useful sequences for regulating transcription are enhancers, splicing signals, transcription termination signals, polyadenylation sites, buffering sequences, RNA processing sequences and other sequences that regulate transgene expression.

[0254] Preferably, the expression system or construct comprises a 3' untranslated region downstream of the nucleotide sequence encoding the desired recombinant protein. This region can increase the expression of the transgene. In this regard, a useful 3' untranslated region is a sequence that provides a poly A signal.

[0255] Suitable heterologous 3'-untranslated sequences can be derived from, for example, SV40 small t antigen, casein 3' untranslated region, or other 3' untranslated sequences well known in the art. Ribosome binding sites are also important for increasing the expression efficiency of FVIII. Similarly, sequences that regulate post-translational modification of FVIII are useful in the present invention.

[0256] Having described the present invention, the present invention will be explained in more detail in the following examples, which are included herein for illustrative purposes only and are not intended to limit the present invention.

[0257] Example 1

[0258] Synthetic liver-specific promoter

[0259] We designed and fully synthesized a number of artificial promoters containing conserved basal promoter elements and transcription start sites. The basal promoter was linked to a number of liver-specific transcription factor binding sites at its 5' end for liver-specific expression. Figure 1 ) (SEQ ID NO: 1) because of its small size (200 bp) and high activity in the human hepatoma cell line Huh7 in vitro using a luciferase reporter gene and transfection experiments.

[0260] The LXP3.3 promoter was then further tested with a LacZ reporter gene packaged in an AAV9 vector that has broad tissue tropism in the liver, heart, and muscle, among others. Parallel in vivo experiments were performed to compare promoter activity and liver specificity with the strong and liver-specific promoter thyroxine binding globulin (TBG), which was previously reported as one of the strongest liver-specific promoters in both small and large animal models. AAV9 vectors containing either LXP3.3-LacZ or TBG-LacZ expression cassettes were injected into C56 / B6 mice via the tail vein at two different doses. Figure 2AAs shown, X-gal staining of liver and heart showed robust hepatic expression and lack of cardiac expression for both promoters. Quantitative analysis of tissue homogenates showed that although the LXP3.3 promoter is only 200 bp in size and the TBG promoter is 681 bp in size, both promoters achieved nearly identical LacZ expression levels and tissue specificity for the liver ( Figure 2B Specifically, quantitative LacZ enzyme activity analysis showed that for the LXP3.3 and TBG promoters, gene expression in the liver was greater than 300-fold higher than in the heart. Furthermore, in the liver, LacZ enzyme activity obtained by the liver-specific promoter was 500-fold higher than that obtained by the ubiquitous CMV promoter ( Figure 2C On the other hand, in the heart, the CMV promoter achieved nearly 13-fold higher LacZ expression than the liver-specific promoter ( Figure 2B ). The results showed that the synthetic promoter LXP3.3 was highly active and specific in the liver.

[0261] Example 2

[0262] Synthetic promoter intron cassette

[0263] The fully synthetic promoter LXP3.3 is linked at its 3' end to a small intron of VH4 with a short non-native exon junction sequence. This intron was initially tested by in vitro transfection experiments using a weak promoter driving the BDD human FVIII gene. The addition of the VH4 intron provides higher gene expression than promoters without introns, and also provides higher expression than the commonly used chimeric intron CIN (Promega) ( Figure 3 ). Therefore, we combined the LXP3.3 promoter and VH4 intron with an artificial exon junction sequence and named it LXP3.3I (SEQ ID NO:2). To test its activity in driving FVIII expression, we inserted the promoter LXP3.3I upstream of a fully synthetic human BDD-deleted FVIII gene (SEQ ID NO:3), which was followed by a small polyadenylation site. Subsequently, the entire gene expression cassette (SEQ ID NO:4) was cloned into an AAV vector plasmid backbone with two AAV inverted terminal repeats for vector DNA replication and vector genome packaging ( Figure 4 ).

[0264] To compare promoter activity in vitro, the ubiquitous CMV promoter linked to the SV40 intron (a powerful combination commonly used) was used to replace LXP3.3I in the FVIII expression cassette. After transfection of the plasmid into the human hepatoma cell line Huh7, FVIII activity was determined using a chromogenic kit. Figure 5As shown in the transfection experiments in , LXP3.3I produces higher FVIII activity in cell culture than the CMV promoter. In addition, we also compared the fully synthetic human BDD-deleted FVIII gene with the BDD FVIII gene of the native human DNA sequence in transfection experiments. When driven by the same LXP3.3I, the two genes encoding the same FVIII amino acid sequence but using different codon usage did not show significant differences in FVIII activity in human cells ( Figure 6 ). However, when a different liver-specific promoter was used, the BDD FVIII gene with native codons showed lower expression compared to the BDD FVIII gene driven by LXP3.3I, indicating that the higher gene expression was mainly due to the LXP3.3I promoter.

[0265] We next tested in vivo gene expression activity in a commonly used FVIII knockout mouse model of hemophilia A. The LXP3.3I-hF8 gene expression cassette was packaged into AAV9 capsids and injected into the tail vein of FVIII KO mice. Two different doses of vector (2 x 10 per mouse) were used. 11 and 4x 10 10 vector genomes) to examine in vivo expression. Fig. 7A As shown, at both doses, high levels and long-term gene expression were achieved for more than 1 year after vector administration. In addition to the chromogenic assay for FVIII activity, an ELISA assay was also used to examine the amount of FVIII protein secreted into plasma ( Figure 7B ). The results show that, compared with the reference standard of full-length wild-type human FVIII protein, BDD FVIII protein concentration is about 50% lower than the reading obtained by the colorimetric determination of FVIII activity relative to the same full-length human FVIII. This difference (ELISA reading lower than FVIII activity reading) may be due to the lack of long B domains in BDD FVIII, which almost contains half of the length of wild-type FVIII. Since the ELISA kit uses a polyclonal antibody for wild-type full-length FVIII including the B domain, it is expected that the BDD FVIII lacking the B domain has fewer antibody binding sites and therefore has a lower ELISA reading. These results show that in hemophilia A mice, both the colorimetric determination and the ELISA determination can produce consistent measurements of FVIII expression. Partial thromboplastin time (PTT) determination was also performed at several time points. The results are basically consistent with those of the other two determination methods.

[0266] Example 3

[0267] Factor VIII heavy chain mutation

[0268] Previous literature has shown that the heavy chain of FVIII is a limiting factor. The heavy chain is much less efficiently processed and / or more unstable than the light chain, and the precise mechanism remains unclear. For example, when the heavy and light chain genes are expressed separately from two separate vectors, a much higher copy number of the heavy chain gene than the light chain gene is required to obtain a protein concentration with similar coagulation activity. Therefore, we set out to improve the post-translational processing efficiency of the FVIII heavy chain by introducing mutations in its C-terminal region, in particular by introducing new glycosylation sites, because glycosylation is well known for its role in post-translational processing and stability of cell membrane-associated and secreted proteins. We chose to introduce glycosylation at the C-terminus of the heavy chain. The rationale is that, as shown in the X-ray crystal structure analysis of the BDDFVIII protein, this terminal region is amorphous and therefore a flexible region without a defined structure. Therefore, mutations in this region may not disrupt the functional structure of the FVIII protein. Based on this rationale, we utilized the two native asparagines at positions 734 and 735 and mutated the adjacent amino acids 736 and 737 to threonine (NNAI to NNTT). The mutations minimized the change in the amino acid sequence and maximized the degree of glycosylation, resulting in two de novo glycosylation sites (NNT and NTT) in the mutant FVIII designated X0 ( Figure 8 ).

[0269] In addition to the two new glycosylation sites in mutant X0, we added another glycosylation site next to the NNTT site by replacing the peptide (EPRSF) at amino acids 738-742 at the N-terminus in the heavy chain (with a native glycosylation sequence (YVNRSL) isolated from amino acids 237 to 242). This native glycosylation site (NRS) was chosen because it is one of the most efficient glycosylation sites in the heavy chain (Medzihradszky et al., Anal. Chem. 69:3986 (1997)), generating a mutant FVIII named X1 ( Figure 8 ).

[0270] Alternatively, we have substituted amino acids EPR at positions 738-740 with NNT in mutant X0, resulting in two additional glycosylation sites in mutant X2 ( Figure 8 ).

[0271] Example 4

[0272] Mutations at the C-terminus of the factor VIII heavy chain enhance the in vitro activity of FVIII

[0273] Next, we compared the FVIII activity of the mutant constructs in parallel with their wild-type BDD FVIII counterparts. Plasmid transfection was performed in the human hepatoma cell line Huh7, which has no endogenous FVIII expression but is amenable to gene expression controlled by a liver-specific promoter. Fig.9A As shown, mutations X1 and X2 more than doubled FVIII activity in vitro compared to the parental gene.

[0274] Example 5

[0275] Long-term and high-level gene expression of mutant FVIII in vivo in a mouse model of hemophilia A

[0276] Since FVIII mutants X1 and X2 showed significantly higher FVIII activity in in vitro transfection experiments, we next examined their gene expression and FVIII activity in FVIII KO mice. The X1 and X2 genes were under the transcriptional control of the same LXP3.3I promoter and were packaged in AAV8 vector particles. Based on earlier experiments, a standard vector dose of 5X 10 10 vg / mice and injected intravenously into hemophilia A mice aged 2 to 3 months via the tail vein. Plasma samples were collected by retro-orbital bleeding, which is a commonly used method. Fig. 9B As shown, mutants X1 and X2 achieved higher expression than their wild-type parent BDD FVIII gene (compared to Fig. 7A To monitor long-term gene expression, we maintained hemophilia A mice treated with either the X1 or X2 mutants for 24 weeks. Similar to hemophilia A mice treated with the parental wt BDDFVIII vector ( Fig. 7A ), mice treated with mutant FVIII vectors also showed long-term stable gene expression without significant reduction in human FVIII activity in plasma. Inhibitor testing at different time points showed no inhibitor formation.

[0277] Those skilled in the art will appreciate that many and various modifications can be made without departing from the spirit of the present invention. Therefore, it should be clearly understood that the form of the present invention is only illustrative and is not intended to limit the scope of the present invention.

[0278] All publications, patent applications, patents, patent publications, Sequences and other references identified by database accession numbers are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0279] The foregoing is illustrative of the present invention and should not be construed as limiting the present invention.

[0280] sequence:

[0281] SEQ ID NO: 1LXP3.3 promoter-200 bp

[0282] SEQ ID NO: 2 (LXP3.3I-288 bp containing LXP3.3 promoter and VH4 intron

[0283] SEQ ID NO:3 Synthetic human B domain deleted factor FVIII coding sequence - 4374 bp

[0284] SEQ ID NO:4 Human factor FVIII gene expression cassette in AAV vector, from left inverted repeat to right inverted terminal repeat - 5045 bp

[0285] SEQ ID NO:5 Human factor FVIII wild type protein sequence without signal peptide - 2333aa. Sequence Listing <110> The University of North Carolina at Chapel Hill Xiao Xiao Li, Juan Yuan Zhenhua <120> OPTIMIZED HUMAN CLOTTINGFACTOR VIII GENE EXPRESSION CASSETTES AND USES THEREOF AND THEIR USE) <130> 5470-734WO <150> US 62 / 112,901 <151> 2015-02-06 <160> 34 <170> PatentIn version 3.5 <210> 1 <211> 200 <212> DNA <213> Homo sapiens <400> 1 ctgtttactc tggttaattt ttaaaggagg gtaaacagtg cctgaaagct gacctttgcc 60 cacattcctc cggtagacat taacttatta aattgattct gattacaaat ctgacctttg 120 cccccatctc acccagtaac aatgcaagag ttgatgtcag tctataaaaa gcgaagcgcg 180 cggtgggcgg ggttcgctgc 200 <210> 2 <211> 288 <212> DNA <213> Artificial <220> <223> LXP3.3I synthetic promoter sequence <400> 2 ctgtttactc tggttaattt ttaaaggagg gtaaacagtg cctgaaagct gacctttgcc 60 cacattcctc cggtagacat taacttatta aattgattct gattacaaat ctgacctttg 120 cccccatctc acccagtaac aatgcaagag ttgatgtcag tctataaaaa gcgaagcgcg 180 cggtgggcgg ggttcgctgc ctgcaggtga gtatctcagg gatccagaca tggggatatg 240 ggaggtgcct ctgatcccag ggctcactgt gggtctctct gttcacag 288 <210> 3 <211> 4374 <212> DNA <213> Artificial <220> <223> Synthetic human B-domain deleted factor FVIII coding sequence <400> 3 atgcagatcg agctgtctac ctgcttcttc ctgtgcctgc tgcggttctg cttcagcgcc 60 accagacggt actatctggg cgccgtggaa ctgagctgggg actacatgca gagcgacctg 120 ggcgagctgc ccgtggatgc cagattccct ccaagagtgc ccaagagctt ccccttcaac 180 acctccgtgg tgtacaagaa aaccctgttc gtggaattca ccgaccacct gttcaatatc 240 gccaagccca gacccccctg gatgggcctg ctgggaccta caattcaggc cgaggtgtac 300 gacaccgtcg tgatcaccct gaagaacatg gccagccacc ccgtgtctct gcatgccgtg 360 ggagtgtcct actggaaggc ctctgagggc gccgagtacg acgatcagac cagccagcgc 420 gagaagagg acgacaaggt gttccctggc ggcagccaca cctacgtgtg gcaggtgctg 480 aaagaaaacg gccccatggc ctccgaccct ctgtgcctga catacagcta cctgagccac 540 gtggacctcg tgaaggacct gaacagcggc ctgatcggag ccctgctcgt gtgtagagag 600 ggcagcctgg ccaaagagaa aacccagacc ctgcacaagt tcatcctgct gttcgccgtg 660 ttcgacgagg gcaagagctg gcacagcgag aaaagaaca gcctgatgca ggaccgggac 720 gccgcctctg ctagagcctg gcccaaaatg cacaccgtga acggctacgt gaacagaagc 780 ctgcccggac tgatcggctg ccaccggaag tctgtgtact ggcacgtgat cggcatgggc 840 accacccctg aggtgcacag catctttctg gaaggacaca cctttctcgt gcggaaccac 900 cggcaggcca gcctggaaat cagccctatc accttcctga ccgcccagac actgctgatg 960 gacctgggcc agtttctgct gttctgccac atcagctccc accagcacga cggcatggaa 1020 gcctacgtga aggtggacag ctgccccgag gaaccccagc tgcggatgaa gaacaacgag 1080 gaagccgagg actacgacga cgacctgacc gacagcgaga tggacgtggt gcgcttcgac 1140 gacgataaca gccccagctt catccagatc agaagcgtgg ccaagaagca ccccaagacc 1200 tgggtgcact atatcgccgc cgaggaagag gactgggatt acgcccctct ggtgctggcc 1260 cccgacgaca gaagctacaa gagccagtac ctgaacaatg gcccccagcg gatcggccgg 1320 aagtataaga aagtgcggtt catggcctac accgacgaga cattcaagac cagagaggcc 1380 atccagcacg agagcggcat cctgggccct ctgctgtatg gcgaagtggg cgacaccctg 1440 ctgatcatct tcaagaacca ggccagcaga ccctacaaca tctaccctca cggcatcacc 1500 gacgtgcggc ccctgtactc tagaaggctg cccaagggcg tgaaacacct gaaggacttc 1560 cccatcctgc ccggcgagat tttcaagtac aagtggaccg tgaccgtgga agatggcccc 1620 accaagagcg accccagatg cctgacacgg tactacagca gcttcgtgaa catggaacgg 1680 gacctggcct ccggcctgat tggcccactg ctgatctgct acaaagaaag cgtggaccag 1740 cggggcaacc agatcatgag cgacaagcgg aacgtgatcc tgtttagcgt gttcgatgag 1800 aaccggtcct ggtatctgac cgagaatatc cagcggttcc tgcccaaccc tgccggcgtg 1860 cagctgggaag atcctgagtt ccaggcctcc aacatcatgc actccatcaa tggctatgtg 1920 ttcgacagcc tgcagctgag cgtgtgcctg cacgaggtgg cctactggta catcctgagc 1980 atcggggccc agaccgactt cctgtccgtg ttcttctccg gctacacctt caagcacaag 2040 atggtgtacg aggataccct gaccctgttc ccctttagcg gcgaaaccgt gttcatgagc 2100 atggaaaacc ccggcctgtg gatcctgggc tgccacaaca gcgacttccg gaacagaggc 2160 atgaccgccc tgctgaaggt gtccagctgc cavagaaca ccggcgacta ctacgaggac 2220 agctatgagg acatcagcgc ctacctgctg agcaagaaca atgccatcga gcccagaagc 2280 ttcagccagc cccctgtgct gaagcggcac cagagagaga tcacccggac caccctgcag 2340 tccgaccagg agagatcga tccgacgac accatcagcg tggaatgaa gaagaagat 2400 ttcgacatct acgacgagga cgagaaccag agcccccggt ccttcagaa aaagacccgg 2460 cacacttca ttgccgctgt ggaacggctg tgggactacg gcatgagcag cagccctcac 2520 gtgctgagaa acagggccca gagcggcagc gtgccccagt tcaagaagt ggtgttccag 2580 gattcacag acggcagctt tacccagcct ctgtaccgcg gcgagctgaa cgaacacctg 2640 ggactgctgg gccctatat ccggccgaa gtggaagata acatcatggt caccttccgg 2700 aatcaggcct cccggcccta cagctctac agctccctga tcagctacga agaggaccag 2760 agacagggcg ctgagccccg gagaacttc gtgaagccca acgagacta gacctacttt 2820 tggaagtgc agcaccacat ggcccctaca aaggacgagt tcgactgca ggcctgggcc 2880 tacttctccg atgtggacct ggaaaaggac gtgcactctg ggctgatcgg ccccctgctc 2940 gtgtgccaca ccaacaccct gaatcccgcc cacggcagac aagtgacagt gcaggaattc 3000 gccctgttct tcaccatctt cgacgaaaca aagagctggt acttcaccga aaacatggaa 3060 agaaactgcc gggctccctg caacatccag atggaagatc ccaccttcaa agagaactac 3120 cggttccacg ccatcaacgg ctacatcatg gacacactgc ccggcctcgt gatggctcag 3180 gatcagcgga tccggtggta tctgctgtcc atgggctcca acgagaacat ccacagcatc 3240 cacttcagcg gccacgtgtt caccgtgcgg aaaaaagaag agtacaaaat ggccctgtac 3300 aacctgtacc ctggggtgtt cgagacagtg gaaatgctgc ccagcaaggc cggcatctgg 3360 cgggtggaat gtctgatcgg cgagcatctg cacgctggga tgagcacact gtttctggtg 3420 tacagcaaca agtgccagac acctctgggc atggcctctg gccacatccg ggactttcag 3480 atcacagcca gcggccagta tggccagtgg gccccaaaac tggccagact gcactacagc 3540 ggcagcatca acgcctggtc caccaaagag cccttcagct ggatcaaggt ggacctgctg 3600 gctcccatga tcatccacgg aatcaagacc cagggcgcca gacagaagtt ctccagcctg 3660 tacatctccc agttcatcat catgtactcc ctggacggca agaagtggca gacctaccgg 3720 ggcaatagca ccggcaccct gatggtgttc ttcggcaacg tggactccag cggcattaag 3780 cacaacatct tcaacccccc catcattgcc cggtacatcc ggctgcaccc cacccactac 3840 agcatccggt ccaccctgag aatggaactg atgggctgcg acctgaactc ctgcagcatg 3900 cccctgggga tggaaagcaa ggccatctcc gacgcccaga tcaccgcctc cagctacttc 3960 accaacatgt tcgccacctg gtccccatcc aaggcccggc tgcatctgca gggcagaagc 4020 aatgcttgga ggccccaagt gaacaacccc aaagaatggc tgcaggtgga cttccagaaa 4080 accatgaaag tgaccggcgt gaccacccag ggcgtgaagt ctctgctgac ctctatgtac 4140 gtgaaagagt tcctgatctc cagcagccag gacggccacc agtggaccct gtttttccag 4200 aacggcaaag tgaaagtgtt tcaggggaac caggacagct tcacccccgt cgtgaatagc 4260 ctggaccctc cactgctgac cagatacctg cggatccacc ctcagagttg ggtgcaccag 4320 attgctctgc ggatggaagt gctgggatgc gaggcccagg acctgtactg ataa 4374 <210> 4 <211> 5045 <212> DNA <213> artificial <220> <223> Recombinant AAV vector sequence <400> 4 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120 gccaactcca tcactagggg ttcctagatc tacgcgtctg tttactctgg ttaattttta 180 aaggagggta aacagtgcct gaaagctgac ctttgcccac attcctccgg tagacattaa 240 cttattaaat tgattctgat tacaaatctg acctttgccc ccatctcacc cagtaacaat 300 gcaagagttg atgtcagtct ataaaaagcg aagcgcgcgg tgggcggggt tcgctgcctg 360 caggtgagta tctcagggat ccagacatgg ggatatggga ggtgcctctg atcccagggc 420 tcactgtggg tctctctgtt cacagcctgc tagcgccacc atgcagatcg agctgtctac 480 ctgcttcttc ctgtgcctgc tgcggttctg cttcagcgcc accagacggt actatctggg 540 cgccgtggaa ctgagctgggg actacatgca gagcgacctg ggcgagctgc ccgtggatgc 600 cagattccct ccaagagtgc ccaagagctt ccccttcaac acctccgtgg tgtacaagaa 660 aaccctgttc gtggaattca ccgaccacct gttcaatatc gccaagccca gacccccctg 720 gatgggcctg ctgggaccta caattcaggc cgaggtgtac gacaccgtcg tgatcaccct 780 gaagacatg gccagccacc ccgtgtctct gcatgccgtg ggagtgtcct actggaaggc 840 ctctgagggc gccgagtacg acgatcagac cagccagcgc gagaaagagg acgacaaggt 900 gttccctggc ggcagccaca cctacgtgtg gcaggtgctg aaagaaaacg gccccatggc 960 ctccgaccct ctgtgcctga catacagcta cctgagccac gtggacctcg tgaaggacct 1020 gaacagcggc ctgatcggag ccctgctcgt gtgtagagag ggcagcctgg ccaaagagaa 1080 aacccagacc ctgcacaagt tcatcctgct gttcgccgtg ttcgacgagg gcaagagctg 1140 gcacagcgag aaagaaca gcctgatgca gcaccgggac gccgcctctg ctagagcctg 1200 gcccaaaatg cacaccgtga acggctacgt gaagaaagc ctgcccggac tgatcggctg 1260 ccaccggaag tctgtgtact ggcacgtgat cggcatgggc accacccctg aggtgcacag 1320 catctttctg gaaggacaca cctttctcgt gcggaaccac cggcaggcca gcctggaaat 1380 cagccctatc accttcctga ccgcccagac actgctgatg gacctgggcc agtttctgct 1440 gttctgccac atcagctccc accagcacga cggcatggaa gcctacgtga aggtggacag 1500 ctgccccgag gaaccccagc tgcggatgaa gaacaacgag gaagccgagg actacgacga 1560 cgacctgacc gacagcgaga tggacgtggt gcgcttcgac gacgataaca gccccagctt 1620 catccagatc agaagcgtgg ccaagaagca ccccaagacc tgggtgcact atatcgccgc 1680 cgaggaagag gactgggatt acgcccctct ggtgctggcc cccgacgaca gaagctacaa 1740 gagccagtac ctgaacaatg gcccccagcg gatcggccgg aagtataaga aagtgcggtt 1800 catggcctac accgacgaga cattcaagac cagagaggcc atccagcacg agagcggcat 1860 cctgggccct ctgctgtatg gcgaagtggg cgacaccctg ctgatcatct tcaagaacca 1920 ggccagcaga ccctacaaca tctaccctca cggcatcacc gacgtgcggc ccctgtactc 1980 tagaaggctg cccaagggcg tgaaacacct gaaggacttc cccatcctgc ccggcgagat 2040 tttcaagtac aagtggaccg tgaccgtgga agatggcccc accaagagcg accccagatg 2100 cctgacacgg tactacagca gcttcgtgaa catggaacgg gacctggcct ccggcctgat 2160 tggcccactg ctgatctgct acaaagaaag cgtggaccag cggggcaacc agatcatgag 2220 cgacaagcgg aacgtgatcc tgtttagcgt gttcgatgag aaccggtcct ggtatctgac 2280 cgagaatatc cagcggttcc tgcccaaccc tgccggcgtg cagctgggaag atcctgagtt 2340 ccaggcctcc aacatcatgc actccatcaa tggctatgtg ttcgacagcc tgcagctgag 2400 cgtgtgcctg cacgaggtgg cctactggta catcctgagc atcggggccc agaccgactt 2460 cctgtccgtg ttcttctccg gctacacctt caagcacaag atggtgtacg aggataccct 2520 gaccctgttc ccctttagcg gcgaaaccgt gttcatgagc atggaaaacc ccggcctgtg 2580 gatcctgggc tgccacaaca gcgacttccg gaacagaggc atgaccgccc tgctgaaggt 2640 gtccagctgc gacaagaaca ccggcgacta ctacgaggac agctatgagg acatcagcgc 2700 ctacctgctg agcaagaca atgccatcga gcccagaagc ttcagccagc cccctgtgct 2760 gaagcggcac cagagagaga tcaccggac caccctgcag tccgaccagg aagagatcga 2820 squirrel squirrel squirrel squirrel 2880 cgagaaccag agccccggt ccttcagaa aaagacccgg cactactca ttgccgctgt 2940 ggaacggctg tgggactacg gcatgagcag cagccctcac gtgctgagaa acagggccca 3000 gagcggcagc gtgccccagt tcaagaagt ggtgttccag gattcacag acggcagctt 3060 tacccagcct ctgtaccgcg gcgagctgaa cgaacacctg ggactgctg gccctatat 3120 ccggccgaa gtggaagata acatcatggt caccttccgg aatcaggcct cccggcccta 3180 cagctctac agctccctga tcagctacga agaggaccag agacaggggcg ctgagccccg 3240 gaagaacttc gtgaagccca acgagactaa gacctacttt tggaggtgc agcaccacat 3300 ggcccctaca aaggacgagt tcgactgcaa ggcctgggcc tactctccg atgtggacct 3360 ggaaaaggac gtgcactctg ggctgatcgg ccccctc gtgtgccaca cacaccct 3420 gatcccgcc cacggcagac aagtgacagt gcaggaattc gccctgttct tcaccattct 3480 cgacgaaaca agagctggt acttcaccga aaacatggaa agaactgcc gggctccctg 3540 ccacatccag atggagatc ccaccttcaag agagaactac cggttccacg ccacaacgg 3600 ctacatcatg gandacactgc ccggcctcgt gatggctcag gatcagcgga tccggtggta 3660 tctgctgtcc atgggctcca acgagaacat ccacagcatc cactcagcg gccacgtgtt 3720 caccgtgcgg aaaaagaag agtacaaat ggccctgtac aacctgtacc ctggggtgtt 3780 cgagacagtg gaatgctgc ccagcaggc cggcatctgg cgggtggaat gtctgatcgg 3840 cgagcatctg cacgctggga tgagcacact gtttctggtg tacagcaca agtgccagac 3900 acctctgggc atggctctg gccacatccg ggactttcag atcacagcca gcggccagta 3960 tggccagtgg gccccaaaac tggccagact gcactacagc ggcagcatca acgcctggtc 4020 caccaaagg cccttcagct ggatcaagt gacctgctg gctcccatga tcatccacgg 4080 aatcaagacc cagggcgcca gagaagtt ctccagcctg tacatctccc agttcatcat 4140 catgtactcc ctggacggca agaagtggca gacctaccgg ggcaatagca ccggcaccct 4200 gatggtgttc ttcggcaacg tggactccag cggcattaag cacaacatct tcaacccccc 4260 catcattgcc cggtacatcc ggctgcaccc cacccactac agcatccggt ccaccctgag 4320 aatggaactg atgggctgcg acctgaactc ctgcagcatg cccctgggga tggaaagcaa 4380 ggccatctcc gacgcccaga tcaccgcctc cagctacttc accaacatgt tcgccacctg 4440 gtccccatcc aaggcccggc tgcatctgca gggcagaagc aatgcttgga ggccccaagt 4500 gaacaacccc aaagaatggc tgcaggtgga cttccagaaa accatgaaag tgaccggcgt 4560 gaccacccag ggcgtgaagt ctctgctgac ctctatgtac gtgaaagagt tcctgatctc 4620 cagcagccag gacggccacc agtggaccct gtttttccag aacggcaaag tgaaagtgtt 4680 tcaggggaac caggacagct tcacccccgt cgtgaatagc ctggaccctc cactgctgac 4740 cagatacctg cggatccacc ctcagagttg ggtgcaccag attgctctgc ggatggaagt 4800 gctgggatgc gaggcccagg acctgtactg ataagtcgac aggcctaata aagagctcag 4860 atgcatcgat cagagtgtgt tggttttttg tgtgagatct aggaacccct agtgatggag 4920 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg 4980 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg 5040 gccaa 5045 <210> 5 <211> 2333 <212> PRT <213> Homo sapiens <400> 5 Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro 50 55 60 Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val 65 70 75 80 Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95 Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110 Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125 Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140 Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175 Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190 His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205 His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg 225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270 Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285 Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300 Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met 305 310 315 320 Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335 Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350 Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365 Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380 Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 385 390 395 400 Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415 Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430 Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445 Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460 Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 465 470 475 480 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495 His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510 Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525 Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540 Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 545 550 555 560 Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575 Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590 Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605 Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620 Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 625 630 635 640 Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655 Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670 Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685 Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700 Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710 715 720 Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735 Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg 740 745 750 Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 755 760 765 Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn 770 775 780 Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro 785 790 795 800 His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815 Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830 Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val 835 840 845 Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860 Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser 865 870 875 880 Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala 885 890 895 Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 900 905 910 Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925 Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940 Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp 945 950 955 960 Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 965 970 975 Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 985 990 Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995 1000 1005 Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu 1010 1015 1020 Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu 1025 1030 1035 Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp 1040 1045 1050 Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr 1055 1060 1065 Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly 1070 1075 1080 Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys 1085 1090 1095 Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His 1100 1105 1110 Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln 1115 1120 1125 Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe 1130 1135 1140 Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr 1145 1150 1155 Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn 1160 1165 1170 Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His 1175 1180 1185 Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr 1190 1195 1200 Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr Val Thr 1205 1210 1215 Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg 1220 1225 1230 Gln Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu 1235 1240 1245 Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys 1250 1255 1260 His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu 1265 1270 1275 Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys 1280 1285 1290 Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr 1295 1300 1305 Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu 1310 1315 1320 Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr 1325 1330 1335 Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr 1340 1345 1350 Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser 1355 1360 1365 Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala 1370 1375 1380 Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser 1385 1390 1395 Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser 1400 1405 1410 Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val 1415 1420 1425 Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu 1430 1435 1440 Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln Arg Glu 1445 1450 1455 Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys 1460 1465 1470 Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485 Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys 1490 1495 1500 Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu 1505 1510 1515 Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile 1520 1525 1530 Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg 1535 1540 1545 Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp 1550 1555 1560 Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu 1565 1570 1575 Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys 1580 1585 1590 Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His 1595 1600 1605 Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu 1610 1615 1620 Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln 1625 1630 1635 Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr 1640 1645 1650 Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1655 1660 1665 Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1670 1675 1680 Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr 1685 1690 1695 Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser 1700 1705 1710 Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1715 1720 1725 Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe 1730 1735 1740 Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 1745 1750 1755 Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 1760 1765 1770 Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1775 1780 1785 Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1790 1795 1800 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 1805 1810 1815 Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asx Asp Cys 1820 1825 1830 Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val 1835 1840 1845 His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr 1850 1855 1860 Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala 1865 1870 1875 Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr 1880 1885 1890 Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met 1895 1900 1905 Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn 1910 1915 1920 Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp 1925 1930 1935 Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn 1940 1945 1950 Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys 1955 1960 1965 Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val 1970 1975 1980 Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg 1985 1990 1995 Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr 2000 2005 2010 Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met 2015 2020 2025 Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln 2030 2035 2040 Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly 2045 2050 2055 Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys 2060 2065 2070 Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln 2075 2080 2085 Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile 2090 2095 2100 Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly 2105 2110 2115 Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser 2120 2125 2130 Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg 2135 2140 2145 Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu 2150 2155 2160 Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro 2165 2170 2175 Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala 2180 2185 2190 Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys 2195 2200 2205 Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln 2210 2215 2220 Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr 2225 2230 2235 Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu 2240 2245 2250 Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp 2255 2260 2265 Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val 2270 2275 2280 Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu 2285 2290 2295 Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser 2300 2305 2310 Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu 2315 2320 2325 Ala Gln Asp Leu Tyr 2330 <210> 6 <211> 8 <212> PRT <213> Artificial <220> <223> Factor VIII consensus insertion sequence <220> <221> MISC_FEATURE <222> (1)..(2) <223> Xaa can be S or T <220> <221> MISC_FEATURE <222> (7) <223> Xaa can be S or T <400> 6 Xaa Xaa Tyr Val Asn Arg Xaa Leu 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 7 Thr Thr Tyr Val Asn Arg Ser Leu 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 8 Thr Thr Tyr Val Asn Arg Thr Leu 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 9 Thr Ser Tyr Val Asn Arg Ser Leu 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 10 Thr Ser Tyr Val Asn Arg Thr Leu 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 11 Ser Thr Tyr Val Asn Arg Ser Leu 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 12 Ser Thr Tyr Val Asn Arg Thr Leu 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 13 Ser Ser Tyr Val Asn Arg Ser Leu 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 14 Ser Ser Tyr Val Asn Arg Thr Leu 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial <220> <223> Factor VIII consensus insertion sequence <220> <221> MISC_FEATURE <222> (1)..(2) <223> Xaa can be S or T <220> <221> MISC_FEATURE <222> (5) <223> Xaa can be S or T <400> 15 Xaa Xaa Asn Asn Xaa 1 5 <210> 16 <211> 5 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 16 Thr Thr Asn Asn Ser 1 5 <210> 17 <211> 5 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 17 Thr Thr Asn Asn Thr 1 5 <210> 18 <211> 5 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 18 Thr Ser Asn Asn Ser 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 19 Thr Ser Asn Asn Thr 1 5 <210> 20 <211> 5 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> 20 Ser Thr Asn Asn Ser 1 5 <210> twenty one <211> 5 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> twenty one Ser Thr Asn Asn Thr 1 5 <210> twenty two <211> 5 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> twenty two Ser Ser Asn Asn Ser 1 5 <210> twenty three <211> 5 <212> PRT <213> Artificial <220> <223> Factor VIII Insertion Sequence <400> twenty three Ser Ser Asn Asn Thr 1 5 <210> twenty four <211> 7 <212> PRT <213> Artificial <220> <223> O-linked glycosylation site consensus sequence <220> <221> misc_feature <222> (2)..(3) <223> Xaa can be any naturally occurring amino acid <220> <221> MISC_FEATURE <222> (6) <223> Xaa can be S or T <400> twenty four Cys Xaa Xaa Gly Gly Xaa Cys 1 5 <210> 25 <211> 5 <212> PRT <213> Artificial <220> <223> O-linked glycosylation site sequence <220> <221> MISC_FEATURE <222> (4) <223> Xaa can be D or E <400> 25 Asn Ser Thr Xaa Ala 1 5 <210> 26 <211> 5 <212> PRT <213> Artificial <220> <223> O-linked glycosylation site sequence <400> 26 Asn Ile Thr Gln Ser 1 5 <210> 27 <211> 5 <212> PRT <213> Artificial <220> <223> O-linked glycosylation site sequence <400> 27 Gln Ser Thr Gln Ser 1 5 <210> 28 <211> 5 <212> PRT <213> Artificial <220> <223> O-linked glycosylation site sequence <220> <221> MISC_FEATURE <222> (1) <223> Xaa can be D or E <220> <221> MISC_FEATURE <222> (4) <223> Xaa can be R or K <400> 28 Xaa Phe Thr Xaa Val 1 5 <210> 29 <211> 4 <212> PRT <213> Artificial <220> <223> O-linked glycosylation site sequence <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa can be D or E <400> 29 Cys Xaa Ser Asn 1 <210> 30 <211> 5 <212> PRT <213> Artificial <220> <223> O-linked glycosylation site sequence <220> <221> MISC_FEATURE <222> (5) <223> Xaa can be R or K <400> 30 Gly Gly Ser Cys Xaa 1 5 <210> 31 <211> 30 <212> PRT <213> Artificial <220> <223> B domain deleted factor VIII sequence <400> 31 Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro 1 5 10 15 Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr 20 25 30 <210> 32 <211> 30 <212> PRT <213> Artificial <220> <223> Mutated B domain deleted factor VIII sequence <400> 32 Leu Ser Lys Asn Asn Thr Thr Glu Pro Arg Ser Phe Ser Gln Asn Pro 1 5 10 15 Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr 20 25 30 <210> 33 <211> 31 <212> PRT <213> Artificial <220> <223> Mutated B domain deleted factor VIII sequence <400> 33 Leu Ser Lys Asn Asn Thr Thr Tyr Val Asn Arg Ser Leu Ser Gln Asn 1 5 10 15 Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr 20 25 30 <210> 34 <211> 28 <212> PRT <213> Artificial <220> <223> Mutated B domain deleted factor VIII sequence <400> 34 Leu Ser Lys Asn Asn Thr Thr Asn Asn Thr Ser Gln Asn Pro Pro Val 1 5 10 15 Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr 20 25

Claims

1. A modified human Factor VIII polypeptide, which is a wild-type human sequence SEQ ID NO: 5, wherein the amino acid residues in the heavy chain of the wild-type human sequence are modified to create one or more glycosylation sites in amino acid residues 731-740, wherein amino acid residues 736 and 737 of the wild-type human sequence SEQ ID NO: 5 are substituted with amino acid residues TT; and wherein amino acid residues 738-742 of the wild-type human sequence SEQ ID NO: 5 are substituted with amino acid residues YVNRSL, or wherein amino acid residues 738-740 of the wild-type human sequence SEQ ID NO: 5 are substituted with amino acid residues NNT and amino acid residues 741-742 are deleted.

2. The modified human Factor VIII polypeptide of claim 1, further comprising a B domain deletion.

3. A polynucleotide encoding the modified human Factor VIII polypeptide of claim 1 or 2.

4. The polynucleotide according to claim 3, operably linked to a promoter.

5. The polynucleotide according to claim 4, wherein the promoter comprises a polynucleotide comprising a synthetic liver-specific promoter, wherein the synthetic liver-specific promoter is the nucleotide sequence shown in SEQ ID NO:

1.

6. The polynucleotide of claim 5, wherein the synthetic liver-specific promoter is operably linked to an intron.

7. The polynucleotide of claim 6, wherein the intron is from VH4.

8. The polynucleotide according to claim 7, wherein the synthetic liver-specific promoter and the intron together are the nucleotide sequence shown in SEQ ID NO:

2.

9. A vector comprising the polynucleotide of any one of claims 3-8.

10. The vector according to claim 9, wherein the vector is a viral vector. The vector according to claim 10 , wherein the vector is an adeno-associated virus vector.

12. The vector according to claim 11, wherein the adeno-associated virus vector is an AAV8 vector or an AAV9 vector.

13. A transformed cell comprising the polynucleotide of any one of claims 3 to 8 and / or the vector of any one of claims 9 to 12.

14. Use of the modified human factor VIII polypeptide of any one of claims 1-2, the polynucleotide of any one of claims 3-8, the vector of any one of claims 9-12, and / or the transformed cell of claim 13 in the preparation of a medicament for treating hemophilia A or acquired factor VIII deficiency in a subject.

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